Relative Resistance of Primary HIV-1 Isolates to Neutralization by Soluble CD4 ERIC S. DAAR,M.D., LosAnge/es,
Ca/torn/a,
DAVID D. HO, M.D., NewYork, New York
Replication of the human immunodeficiency virus type 1 (HIV-l) underlies the pathogenesis and progression of the acquired immunodeficiency syndrome (AIDS). A soluble form of the virus receptor, CD4, has been developed as a potential therapeutic agent with good activity against laboratory strains of HIV-l in vitro. However, quantitative virologic studies performed to date on the blood of patients receiving recombinant soluble CD4 (sCD4) demonstrated no efficacy in viva despite good drug levels in serum. These results led us to examine the neutralizing activity of sCD4 against multiple primary HIV-1 isolates from infected patients. The findings demonstrate that primary isolates were significantly more resistant to sCD4 than were laboratory strains, which suggests a need to reevaluate CD4-based therapies and to conduct better designed preclinical studies that include experiments performed on patient viral isolates.
A
cquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus type 1 (HIV-l) [l-3]. Treatment of AIDS is based on the premise that continued viral replication underlies both the pathogenesis and progression of the disease. HIV-l replication occurs primarily within CD4+ helper/inducer lymphocytes [4-61 and monocyteimacrophages [7-101. A number of studies have demonstrated that the CD4 molecule is the cellular receptor for HIV-l [11,12]. Subsequently, several investigative teams have genetically engineered truncated and soluble forms of CD4 that have exhibited potent antiviral properties in vitro [13-171. In fact, the 90% inhibitory doses (ID& of recombinant soluble CD4 (sCD4) for laboratory strains of HIV-l range from 0.1 to 1.0 pg. In addition, the simian immunodeficiency virus (SIV) and HIV type 2 (HIV-2) were inhibited by sCD4 [18,191, although the LAV-2uon strain of HIV-2 required higher concentrations of sCD4 for neutralization than did strains of HIV-l previously tested [19]. Furthermore, several investigators have demonstrated significant antiviral activity in vitro with constructs that link sCD4 to immunoglobulins G and M (IgG, IgM) or cytotoxins [20-231. We now outline how drug studies in vitro may be misleading, using clinical and laboratory results generated on sCD4. Specifically, the data show that in vitro experiments that utilize highly passaged HIV-l laboratory strains in T-cell lines are not truly reflective of the situation with primary viral isolates in normal peripheral blood mononuclear cells (PBMCs). Our findings have significant implications for all CD4-based therapies, as well as for the proper conduct of preclinical studies of promising AIDS drugs.
PHASEi/II TRIAL AND EX WU EXPERIMENTS
From the Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, University of California, Los Angeles School of Medicine, Los 1 Angeles, California. Requests for reprints should be addressed to David D. Ho, M.D., Aaron Dlamond AIDS Research Center, New York University School of Medicine, 455 First Avenue, New York, New York 10016.
4A-22s
April 10, 1991
The American Journal of Medicine
In a Phase I/II trial of sCD4 (Biogen) [17], 12 patients with AIDS or AIDS-related complex (ARC) at our hospital received 1, 3, 9, or 30 mg/day of sCD4 for at least 28 days [24]. The toxicity profile and pharmacokinetics of sCD4 have previously been reported [24]. The patients receiving 1, 3, and 9 mg/day demonstrated no clinical benefit or significant decrease in serum p24 core antigen levels.
Volume 90 (suppl 4A)
SYMPOSIUM
ON COMBINATION
ANTIRETROVIRAL
THERAPY / DAAR and HO
80
60
Figure 1. HIV neutralization by sCD4 (Biogen) in vitro. Neutralization of each viral isolate is represented by a computer-generated best-fit curve. HTLV-IIIB and HTLV-IIIRF represent laboratory strains. Isolates: M, HIV-2 isolate LAV-2,oo; Nl, HIV(JR-FL), and N2, HIV(JR-CSF), both molecular clones of primary isolates. The other letters represent once-passaged primary isolates.
0
0.1
1
10
sCD4
Furthermore, in the five patients who received 30 mg/day, there was no consistent decrease in plasma p24 core antigen or in infectious HIV-l titers in plasma or PBMC despite adequate serum sCD4 levels (mean value of 156 ng/mL) [25]. Similarly, little antiviral efficacy in vivo was demonstrated in a Phase I/II trial using another sCD4 preparation (Genentech) [26]. The discrepancy between the in vivo and the in vitro results led to further evaluation of sCD4 in our laboratory. Ex vivo experiments were performed to test the activity of sCD4 to neutralize infection of stimulated normal PBMC by unpassaged (unselected) patient HIV-l isolates in the presence of plasma. Specifically, each of the plasma samples from eight patients with AIDS was divided into equal aliquots and increasing concentrations of sCD4 were added to the aliquots. Each aliquot was subsequently titered by end-point-dilution cultures to determine the concentration of sCD4 required in plasma to decrease the infectious titer. Surprisingly, six of eight plasma samples showed no decrease in titer despite sCD4 concentrations as high as 1.0 mg/mL [25]. Three potential explanations for the refractoriness of primary HIV-l isolates to sCD4 ex vivo were considered. The first possibility was the interference or inactivation of sCD4 by factors present in plasma. In addition, the relative re-
100
1000
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sistance to sCD4 may have been due to the differences in the target cells used in the experiments. Finally, the explanation may lie in intrinsic differences between primary and laboratory HIV-l strains.
PLASMAINTERFERENCEAND TARGET CELLDIFFERENCES Interference or inactivation of sCD4 by plasma was largely excluded by experiments adding heat-inactivated subneutralizing concentrations of HIV-l antibody-positive plasma to known titers of laboratory strains of HIV-l. Neutralization of laboratory viruses by sCD4 in the presence of seropositive plasma was not significantly different from that with virus alone (Daar ES, Ho DD, unpublished data). The discrepancy between the in vitro and ex vivo findings could also be explained by differences between tumor T-cell lines and normal PBMCs. However, in experiments specifically designed to address this issue, there appeared to be no intrinsic difference between the ability of sCD4 to neutralize infection of laboratory strains of HIV-l in T-cell lines and normal PBMCs [25]. In addition, infection of PBMCs by both primary and laboratory HIV-l isolates was easily blocked with low concentrations of the anti-CD4 monoclonal antibody Leu3A, sug-
April 10, 1991
The American Journal of Medicine
Volume 90 (suppl 4A)
4A-23s
SYMPOSIUM
ON COMBINATION
ANTIRETROVIRAL
THERAPY / DAAR and HO
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Figure 2. Neutralization of HIV-1 by multimeric IgM-TA All isolates were 100% neutralized at 100 pg. Isolates: n = HTLV-III@ l = HIV(JRFL); 0 = HIV(JR-CSF); 0 = E; and A = P.
gesting that CD4 is the principal receptor for HIV-l in PBMCs and that the relative resistance of primary HIV-l isolates to sCD4 is not due to a CDCindependent mechanism of virus entry [25]. Furthermore, the infectivity of monocytotropic HIV-l isolates is similarly blocked by Leu3A and sCD4 [25]. In fact, with a given viral isolate, there is little difference in the IDa for sCD4 using lymphocytes or monocytelmacrophages as target cells. These data suggest that the relative resistance to sCD4 observed in viva and ex vivo is most likely the result of specific viral differences.
NEUTRALIZATIONOF PRIMARYISOLATES IN VITRO Two laboratory strains and nine primary isolates of HIV-l derived from the plasma of patients with AIDS or ARC were tested for susceptibility to neutralization with sCD4 in vitro. As shown in Figure 1, the laboratory strains of HIV were readily neutralized at concentrations of less than 1.0 pg, a fmding consistent with previous reports. In contrast, the primary isolates were relatively refractory, with the IDg0 200 to 2,000 times higher than those observed with the laboratory strains. In addition, as demonstrated by other investigators, the LAV2non strain of HIV-2 had an IDa markedly higher than that of human T-cell lymphotropic virus (HTLV)-IIIB (Figure 1) [l&19]. This refractoriness was not specific to any one product, since a third sCD4 (Smith Kline Beecham) yielded virtu4A-24s
April 10, 1991
The American Journal of Medicine
ally identical results against HTLV-IIIB and two primary isolates [25]. Primary HIV-l isolates were also relatively resistant to neutralization by the sCD4 hybrid molecules, IgG-T4 and IgM-T4, provided by A. Traunecker [25]. In fact, IDo,, of the bivalent IgG-T4 molecule for the primary isolates was more than 100 times higher than that for the laboratory strain HTLV-IIIB [25]. Similarly, primary isolates were relatively resistant to the lo-valency IgM-T4 molecule, as shown in Figure 2. What is the HIV-l determinant for sensitivity or resistance to neutralization by sCD4? By use of recombinant viruses, we have recently found that the relative resistance to sCD4 is determined in the env gene. O’Brien eC al [27] have generated many viruses by combining portions of the viral genome from NL4-3 (a laboratory strain) and HIV(JR-FL) (a primary isolate); one recombinant virus is NFNSX, which is composed of the NL4-3 genome except that the env gene is that of HIV(JR-FL). As shown in Figure 3, NFN-SX has similar resistance to sCD4 neutralization, as does HIV(JR-FL). Therefore, expectedly, the susceptibility to sCD4 neutralization is determined by the envelope glycoproteins of the HIV-l isolate. Why are primary HIV-l isolates more resistant to sCD4? Several mechanisms can be postulated, but we have already excluded CDkindependent infection of PBMCs and excessive defective particle formation as potential explanations [25]. One im-
Volume 90 (suppl 4A)
SYMPOSIUM
ON COMBINATION
ANTIRETROVIRAL
THERAPY /DAAR and HO
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60
0 Figure 3. Neutralization isolates
by
sCD4.
0 = HIV(JR-FL); +
of recombinant Isolates:
= NFN-SX.
0.0 1
0.1
1
viral
0 = NL4-3,
sCD4
I
portant remaining possibility is that the gp120 of primary isolates has a lower affinity for CD4. Although the lower affinity may not affect HIV-l infection (which is mediated by multimeric gp120CD4 interactions), it can significantly affect the neutralizing capacity of sCD4. There is indeed some evidence suggesting that variable affinity of gp120CD4 interaction may influence sCD4 susceptibility. For example, the HIV-l isolate Ba-L has a sixfold lower affinity for CD4 and is proportionally more resistant to sCD4 [28]. In addition, the envelope glycoprotein of the HIV-2 strain LAV-2soo has been found to have a 25fold lower affinity for CD4 when compared with that of the HTLV-IIIB isolate [19]. We have found that LAV-2noo is about 170 times more resistant to sCD4 [25]. Another potential explanation is variable ability of sCD4 to induce dissociation of gp120 from the virions of different HIV-1 strains. Recently, Moore et al [29] demonstrated that sCD4 may not only block infection by competing with cellular CD4, but also by stripping gp120 from its complex with gp41. Any strain-to-strain variation in this property could result in altered neutralization characteristics with sCD4.
COMMENT Our data [25], together with that of others using primary isolates in monocytelmacrophages and PBMCs [30], suggest that primary isolates are rela-
10
100
tug)
tively resistant to neutralization by sCD4. Although the precise mechanism for this resistance has not been defined, the observed refractoriness to monomeric sCD4 and multimeric sCD4 constructs in viva, ex vivo, and in vitro represents significant obstacles to CD4-based therapies. These findings reflect the fact that HIV-l exists in vivo as a heterogeneous population of viruses, whose basic virologic properties must be considered when designing and conducting preclinical studies. In fact, our results emphasize the importance of and the need for studying unpassaged HIV-l isolates ex vivo as well as minimally passaged viruses in vitro as a part of routine preclinical testing for all promising anti-HIV agents.
ACKNOWLEDGMENTS We are indebted to Drs. W.A. O’Brien and I.S.Y. Chen for recombinant viruses, and to W. Chen for illustrations.
REFERENCES 1. Barre-Sinoussi F, Chermann JC, Rey F, et al: Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficrency syndrome (AIDS). Science 1983; 220: 868-71. 2. Gallo RC, Salahuddin SZ, Popovic M, et al: Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 1984; 224: 500-3. 3. Popov~c M, Sarngadharan MG, Read E, Gallo RC: Detection, isolatron, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 1984; 224: 497-500. 4. Gottieb MS, Schroff R, Schanker HM, etal: Pneumocystis carinii pneumonia and
April 10, 1991
The American Journal of Medicine
Volume 90 (suppl 4A)
4A-2%
mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency. N Engl J Med 1981; 305: 1425-31. 5. Bowen DL, Lane HC, Fauci AS: lmmunopathogenesis of the acquired immunodeficiency syndrome. Ann Intern Med 1985; 103: 704-9. 6. Klatzmann D, Barre-Sinoussi F, Nugeyre MT, et&Selective tropism of lymphadenopathy-associated virus (LAV) for helper-inducer T lymphocyte. Science 1984; 225: 59-64. 7. Ho DD, Rota TR, Hirsch MS: Infection of monocyte/macrophages by human T lymphotropic virus type Ill. J Clin Invest 1986; n: 1712-5. 8. Nicholson JKA, Gross GD, Callaway CS, McDougal JS: In vitro infection of human monocytes with human T-lymphotropic virus type Ill/lymphadenopathy-associated virus (HTLV III/LAV). J lmmunol 1986; 137: 323-9. 9. Salahuddin SZ, Rose RM, Groopman JE, Markham PD, Gallo RC: Human T lymphotropic virus type Ill infection of human alveolar macrophages. Blood 1986; 68: 281-4. 10. Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, Popovic M: The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 1986; 233: 215-9. 11. Dalgleish AG, Beverly PCL, Clapham PR, Crawford DH, Greaves MF, Weiss RA: The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovi111s.Nature 1984; 312: 763-7. 12. Klatzmann D, Champagne E, Chamaret S, et a/: T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 1984; 312: 767-8. 13. Smith DH, Byrn RA, Marsters SA, Gregory T, Groopman JE, Capon DJ: Blocking of HIV infection by a soluble, secreted form of CD4 antigen. Science 1987; 238: 1704-6. 14. Deen KC, McDougal JS, lnacker R, et al: A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature 1988; 331: 82-4. 15. Traunecker A, Luke W, Karjalaninen K: Soluble CD4 molecule neutralized human immunodeficiency virus type 1. Nature 1988; 331: 84-86. 16. Hussey RE, Richardson NE, Kowalski M, et al: A soluble CD4 protein selectively inhibits HIV replication and syncytium formation. Nature 1988; 331: 78-81. 17. Fisher RA, Bertonis JM, Werner M, et al: HIV infection is blocked in vitro by recombinant soluble CD4. Nature 1988; 331: 76-8. 18. Clapham PR, Weber JN, Whitby D, et at Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T cells and monocytes but not for brain and
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April 10, 1991
The American Journal of Medicine
muscle cells. Nature 1989; 337: 368-70. 19. Moore JP: Simple methods for monitoring HIV-1 and HIV-2 gp120 binding to sCD4 by ELISA: HIV-2 has a 25 fold lower affinity than HIV-1 for sCD4. AIDS 1990; 4: 297-305. 20. Traunecker A, Schneider J, Kiefer H, Karjalainen K: Highly efficient neutralization of HIV with recombinant CD4immunoglobulin molecule. Nature 1989; 339:
68-70. 21. Capon DJ, Chamow SM, Mordenti J, et a6 Designing CD4 immunoadhesins for AIDS therapy. Nature 1989; 337: 525-31. 22. Chaudhary VK, Mizukami T, Fuerst TR, et at Selective killing of HIV-infected cells by recombinant human CD4-pseudomonas exotoxin hybrid protein. Nature 1988; 335: 369-372. 23. Berger EA, Clouse KA, Chaudhary VK, et at CDCPseudomonas exotoxin hybrid protein blocks the spread of human immunodeficiency virus infection in vitro and is active against cells expressing the envelope glycoprotein from diverse primate immunodeficiency retroviruses. Proc Natl Acad Sci USA 1989; 86: 9539-43. 24. Schooley RT, Merigan TC, Gaut P, et a6 A phase l/II escalating dose trial of recombinant soluble CD4 therapy in patients with AIDS and AIDS-related complex. Ann Intern Med 1990; 112: 247-53. 25. Daar ES, Li X-L, Moudgil T, Ho DD: High concentrations of recombinant soluble CD4 are required to neutralize primary human immunodeficiency virus type I isolates Proc Natl Acad Sci USA 1990; 87: 6574-8. 26. Kahn JO, Allan JD, Hodges TL, et at The safety and pharmacokinetics of recombinant soluble CD4 (rCD4) in subjects with the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. Ann Intern Med 1990; 112: 254-61. 27. O’Brien WA, Koyanagi Y, Namazie A, et at HIV-1 tropism for mononuclear phagocytes can be determined by regions of gp120 outside the CDCbinding domain. Nature 1990; 348: 69-73. 28. Ivey-Hoyle M, Culp JS, Chaikin MA, et at Envelope glycoproteins from biologically diverse isolates of immunodeficiency viruses have widely different affinities for CD4. Proc Natl Acad Sci USA 1991; 88: 512-16. 29. Moore JP, McKeating JA, Weiss RA, Sattentau QJ: Dissociation of gp120 from HIV-1 virions induced by soluble CD4. Science 1990; 250: 1139-42. 30. Gomatos PJ, Stamatos NM, Gendelman HE, et al: Relative inefficiency of soluble recombinant CD4 for inhibition of infection by monocyte-tropic HIV in monocytes and T cells. J lmmunol 1990; 144: 4183-8.
Volume 90 (suppl 4A)
Ca/torn/a,
DAVID D. HO, M.D., NewYork, New York
Replication of the human immunodeficiency virus type 1 (HIV-l) underlies the pathogenesis and progression of the acquired immunodeficiency syndrome (AIDS). A soluble form of the virus receptor, CD4, has been developed as a potential therapeutic agent with good activity against laboratory strains of HIV-l in vitro. However, quantitative virologic studies performed to date on the blood of patients receiving recombinant soluble CD4 (sCD4) demonstrated no efficacy in viva despite good drug levels in serum. These results led us to examine the neutralizing activity of sCD4 against multiple primary HIV-1 isolates from infected patients. The findings demonstrate that primary isolates were significantly more resistant to sCD4 than were laboratory strains, which suggests a need to reevaluate CD4-based therapies and to conduct better designed preclinical studies that include experiments performed on patient viral isolates.
A
cquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus type 1 (HIV-l) [l-3]. Treatment of AIDS is based on the premise that continued viral replication underlies both the pathogenesis and progression of the disease. HIV-l replication occurs primarily within CD4+ helper/inducer lymphocytes [4-61 and monocyteimacrophages [7-101. A number of studies have demonstrated that the CD4 molecule is the cellular receptor for HIV-l [11,12]. Subsequently, several investigative teams have genetically engineered truncated and soluble forms of CD4 that have exhibited potent antiviral properties in vitro [13-171. In fact, the 90% inhibitory doses (ID& of recombinant soluble CD4 (sCD4) for laboratory strains of HIV-l range from 0.1 to 1.0 pg. In addition, the simian immunodeficiency virus (SIV) and HIV type 2 (HIV-2) were inhibited by sCD4 [18,191, although the LAV-2uon strain of HIV-2 required higher concentrations of sCD4 for neutralization than did strains of HIV-l previously tested [19]. Furthermore, several investigators have demonstrated significant antiviral activity in vitro with constructs that link sCD4 to immunoglobulins G and M (IgG, IgM) or cytotoxins [20-231. We now outline how drug studies in vitro may be misleading, using clinical and laboratory results generated on sCD4. Specifically, the data show that in vitro experiments that utilize highly passaged HIV-l laboratory strains in T-cell lines are not truly reflective of the situation with primary viral isolates in normal peripheral blood mononuclear cells (PBMCs). Our findings have significant implications for all CD4-based therapies, as well as for the proper conduct of preclinical studies of promising AIDS drugs.
PHASEi/II TRIAL AND EX WU EXPERIMENTS
From the Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, University of California, Los Angeles School of Medicine, Los 1 Angeles, California. Requests for reprints should be addressed to David D. Ho, M.D., Aaron Dlamond AIDS Research Center, New York University School of Medicine, 455 First Avenue, New York, New York 10016.
4A-22s
April 10, 1991
The American Journal of Medicine
In a Phase I/II trial of sCD4 (Biogen) [17], 12 patients with AIDS or AIDS-related complex (ARC) at our hospital received 1, 3, 9, or 30 mg/day of sCD4 for at least 28 days [24]. The toxicity profile and pharmacokinetics of sCD4 have previously been reported [24]. The patients receiving 1, 3, and 9 mg/day demonstrated no clinical benefit or significant decrease in serum p24 core antigen levels.
Volume 90 (suppl 4A)
SYMPOSIUM
ON COMBINATION
ANTIRETROVIRAL
THERAPY / DAAR and HO
80
60
Figure 1. HIV neutralization by sCD4 (Biogen) in vitro. Neutralization of each viral isolate is represented by a computer-generated best-fit curve. HTLV-IIIB and HTLV-IIIRF represent laboratory strains. Isolates: M, HIV-2 isolate LAV-2,oo; Nl, HIV(JR-FL), and N2, HIV(JR-CSF), both molecular clones of primary isolates. The other letters represent once-passaged primary isolates.
0
0.1
1
10
sCD4
Furthermore, in the five patients who received 30 mg/day, there was no consistent decrease in plasma p24 core antigen or in infectious HIV-l titers in plasma or PBMC despite adequate serum sCD4 levels (mean value of 156 ng/mL) [25]. Similarly, little antiviral efficacy in vivo was demonstrated in a Phase I/II trial using another sCD4 preparation (Genentech) [26]. The discrepancy between the in vivo and the in vitro results led to further evaluation of sCD4 in our laboratory. Ex vivo experiments were performed to test the activity of sCD4 to neutralize infection of stimulated normal PBMC by unpassaged (unselected) patient HIV-l isolates in the presence of plasma. Specifically, each of the plasma samples from eight patients with AIDS was divided into equal aliquots and increasing concentrations of sCD4 were added to the aliquots. Each aliquot was subsequently titered by end-point-dilution cultures to determine the concentration of sCD4 required in plasma to decrease the infectious titer. Surprisingly, six of eight plasma samples showed no decrease in titer despite sCD4 concentrations as high as 1.0 mg/mL [25]. Three potential explanations for the refractoriness of primary HIV-l isolates to sCD4 ex vivo were considered. The first possibility was the interference or inactivation of sCD4 by factors present in plasma. In addition, the relative re-
100
1000
tug)
sistance to sCD4 may have been due to the differences in the target cells used in the experiments. Finally, the explanation may lie in intrinsic differences between primary and laboratory HIV-l strains.
PLASMAINTERFERENCEAND TARGET CELLDIFFERENCES Interference or inactivation of sCD4 by plasma was largely excluded by experiments adding heat-inactivated subneutralizing concentrations of HIV-l antibody-positive plasma to known titers of laboratory strains of HIV-l. Neutralization of laboratory viruses by sCD4 in the presence of seropositive plasma was not significantly different from that with virus alone (Daar ES, Ho DD, unpublished data). The discrepancy between the in vitro and ex vivo findings could also be explained by differences between tumor T-cell lines and normal PBMCs. However, in experiments specifically designed to address this issue, there appeared to be no intrinsic difference between the ability of sCD4 to neutralize infection of laboratory strains of HIV-l in T-cell lines and normal PBMCs [25]. In addition, infection of PBMCs by both primary and laboratory HIV-l isolates was easily blocked with low concentrations of the anti-CD4 monoclonal antibody Leu3A, sug-
April 10, 1991
The American Journal of Medicine
Volume 90 (suppl 4A)
4A-23s
SYMPOSIUM
ON COMBINATION
ANTIRETROVIRAL
THERAPY / DAAR and HO
80
80
Figure 2. Neutralization of HIV-1 by multimeric IgM-TA All isolates were 100% neutralized at 100 pg. Isolates: n = HTLV-III@ l = HIV(JRFL); 0 = HIV(JR-CSF); 0 = E; and A = P.
gesting that CD4 is the principal receptor for HIV-l in PBMCs and that the relative resistance of primary HIV-l isolates to sCD4 is not due to a CDCindependent mechanism of virus entry [25]. Furthermore, the infectivity of monocytotropic HIV-l isolates is similarly blocked by Leu3A and sCD4 [25]. In fact, with a given viral isolate, there is little difference in the IDa for sCD4 using lymphocytes or monocytelmacrophages as target cells. These data suggest that the relative resistance to sCD4 observed in viva and ex vivo is most likely the result of specific viral differences.
NEUTRALIZATIONOF PRIMARYISOLATES IN VITRO Two laboratory strains and nine primary isolates of HIV-l derived from the plasma of patients with AIDS or ARC were tested for susceptibility to neutralization with sCD4 in vitro. As shown in Figure 1, the laboratory strains of HIV were readily neutralized at concentrations of less than 1.0 pg, a fmding consistent with previous reports. In contrast, the primary isolates were relatively refractory, with the IDg0 200 to 2,000 times higher than those observed with the laboratory strains. In addition, as demonstrated by other investigators, the LAV2non strain of HIV-2 had an IDa markedly higher than that of human T-cell lymphotropic virus (HTLV)-IIIB (Figure 1) [l&19]. This refractoriness was not specific to any one product, since a third sCD4 (Smith Kline Beecham) yielded virtu4A-24s
April 10, 1991
The American Journal of Medicine
ally identical results against HTLV-IIIB and two primary isolates [25]. Primary HIV-l isolates were also relatively resistant to neutralization by the sCD4 hybrid molecules, IgG-T4 and IgM-T4, provided by A. Traunecker [25]. In fact, IDo,, of the bivalent IgG-T4 molecule for the primary isolates was more than 100 times higher than that for the laboratory strain HTLV-IIIB [25]. Similarly, primary isolates were relatively resistant to the lo-valency IgM-T4 molecule, as shown in Figure 2. What is the HIV-l determinant for sensitivity or resistance to neutralization by sCD4? By use of recombinant viruses, we have recently found that the relative resistance to sCD4 is determined in the env gene. O’Brien eC al [27] have generated many viruses by combining portions of the viral genome from NL4-3 (a laboratory strain) and HIV(JR-FL) (a primary isolate); one recombinant virus is NFNSX, which is composed of the NL4-3 genome except that the env gene is that of HIV(JR-FL). As shown in Figure 3, NFN-SX has similar resistance to sCD4 neutralization, as does HIV(JR-FL). Therefore, expectedly, the susceptibility to sCD4 neutralization is determined by the envelope glycoproteins of the HIV-l isolate. Why are primary HIV-l isolates more resistant to sCD4? Several mechanisms can be postulated, but we have already excluded CDkindependent infection of PBMCs and excessive defective particle formation as potential explanations [25]. One im-
Volume 90 (suppl 4A)
SYMPOSIUM
ON COMBINATION
ANTIRETROVIRAL
THERAPY /DAAR and HO
80
60
0 Figure 3. Neutralization isolates
by
sCD4.
0 = HIV(JR-FL); +
of recombinant Isolates:
= NFN-SX.
0.0 1
0.1
1
viral
0 = NL4-3,
sCD4
I
portant remaining possibility is that the gp120 of primary isolates has a lower affinity for CD4. Although the lower affinity may not affect HIV-l infection (which is mediated by multimeric gp120CD4 interactions), it can significantly affect the neutralizing capacity of sCD4. There is indeed some evidence suggesting that variable affinity of gp120CD4 interaction may influence sCD4 susceptibility. For example, the HIV-l isolate Ba-L has a sixfold lower affinity for CD4 and is proportionally more resistant to sCD4 [28]. In addition, the envelope glycoprotein of the HIV-2 strain LAV-2soo has been found to have a 25fold lower affinity for CD4 when compared with that of the HTLV-IIIB isolate [19]. We have found that LAV-2noo is about 170 times more resistant to sCD4 [25]. Another potential explanation is variable ability of sCD4 to induce dissociation of gp120 from the virions of different HIV-1 strains. Recently, Moore et al [29] demonstrated that sCD4 may not only block infection by competing with cellular CD4, but also by stripping gp120 from its complex with gp41. Any strain-to-strain variation in this property could result in altered neutralization characteristics with sCD4.
COMMENT Our data [25], together with that of others using primary isolates in monocytelmacrophages and PBMCs [30], suggest that primary isolates are rela-
10
100
tug)
tively resistant to neutralization by sCD4. Although the precise mechanism for this resistance has not been defined, the observed refractoriness to monomeric sCD4 and multimeric sCD4 constructs in viva, ex vivo, and in vitro represents significant obstacles to CD4-based therapies. These findings reflect the fact that HIV-l exists in vivo as a heterogeneous population of viruses, whose basic virologic properties must be considered when designing and conducting preclinical studies. In fact, our results emphasize the importance of and the need for studying unpassaged HIV-l isolates ex vivo as well as minimally passaged viruses in vitro as a part of routine preclinical testing for all promising anti-HIV agents.
ACKNOWLEDGMENTS We are indebted to Drs. W.A. O’Brien and I.S.Y. Chen for recombinant viruses, and to W. Chen for illustrations.
REFERENCES 1. Barre-Sinoussi F, Chermann JC, Rey F, et al: Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficrency syndrome (AIDS). Science 1983; 220: 868-71. 2. Gallo RC, Salahuddin SZ, Popovic M, et al: Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 1984; 224: 500-3. 3. Popov~c M, Sarngadharan MG, Read E, Gallo RC: Detection, isolatron, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 1984; 224: 497-500. 4. Gottieb MS, Schroff R, Schanker HM, etal: Pneumocystis carinii pneumonia and
April 10, 1991
The American Journal of Medicine
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