545
Studies of Antibody-Dependent Enhancement of Human Immunodeficiency Virus (HIV) Type 1 Infection Mediated by Fe Receptors Using Sera from Recipients of a Recombinant gp160 Experimental HIV-l Vaccine Richard H. Haubrich,* Akira Takeda, Wayne Koff, Gale Smith, and Francis A. Ennis
University of Massachusetts Medical Center, Worcester; AIDS Vaccine Evaluation Groups, National Institute for Allergy and Infectious Diseases. National Institutes of Health. Bethesda. Maryland; Microgenesys, Inc.. Meriden, Connecticut
Human immunodeficiency virus (HIV) infection is a tremendous global problem. During 1980-1990, > 161 ,000 cases of AIDS were reported in the United States. Intense efforts are underway to develop treatments for HIV and its resultant opportunistic infections. To prevent new HIV infections, a safe, effective vaccine is needed. Until recently, vaccine development was hindered by the inability to show protection from retroviral infections in animals. Three groups have recently reported protection against simian immunodeficiency virus challenge in monkeys [1-3]. A degree of protection against HIV-1 has also been reported in challenged chimpanzees [4]. One theoretical risk of vaccination is that antibodies to HIV might augment infection instead of neutralizing the virus. This augmentation ofinfection by antibodies is termed antibody-dependent enhancement (ADE). ADE was first described in animal viruses by Hawkes [5] and has been characterized most extensively for dengue virus. Studies on ADE in dengue have used convalescent-phase sera. Subneutralizing concentrations of antibody-augmented dengue virus yield 2-
Received 25 July 1991; revised 24 October 1991. Presented in part: VII International Conference on AIDS. June 1991, Florence. Italy (abstract MA 1327). All subjects gave informed consent. Financial support: National Institutes of Health (AI-24750. AI-26458. DAMD-C-279, AI-Onn, AI-25831). Reprints or correspondence: Dr. Francis A. Ennis, University of Massachusetts Medical Center, Division oflnfectious Diseases and Immunology, 55 Lake Ave. N., Worcester, MA 01655. * Present address: University of California San Diego Medical Center. The Journal of Infectious Diseases 1992;165:545-8 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6503-0021 $0 1.00
to 200-fold. The enhancing activity was in the IgG fraction of serum. Complement was not required for enhancement, but the Fe part ofIgG was necessary [6]. HIV is known to infect cells via the CD4 receptor [7]. T cells, rnonocytes, and monocytic cell lines are infected via this receptor. Several studies suggest that CD4 is not the only receptor for HIV. Glial cells, which do not have CD4, and NK cells, which have FcR-III (Fe receptor III ofIgG) but not CD4, have been infected with HIV [8, 9]. There are three possible mechanisms by which HIV could enter monocytic cells: nonspecific phagocytosis, CD4-mediated binding followed by fusion or endocytosis, and endocytosis of HI V-antibody complexes via the FcR ofIgG or the complement receptor. We have shown that HIV infection of the monocytic cell line U937 [10] and human monocytes [11] can be increased by adding subneutralizing concentrations of anti-HlV antibodies. This enhancing activity is present in the IgG fraction of serum and requires the FcR ofIgG and CD4 [11, 12]. The clinical significance of ADE has not been determined. In vitro experiments show that virus yield is increased two- to threefold in the presence oflow concentrations of antibody [10]. This difference is small compared with ADE with dengue in vitro. However, HIV infection lasts several years, and twofold increases in viral yield over thousands of infectious cycles in vivo may increase the viral burden and contribute to the pathogenesis of HI V infection. Although there are no in vivo data that demonstrate a role for ADE, it is considered a theoretical risk of HIV vaccination if the vaccine contains epitopes that induce enhancing antibodies. The epitopes that enhance infection have not been defined, and until the epitopes are mapped, it will be important to test vaccines for ADE. We tested sera from
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 25, 2015
Subneutralizing concentrations of sera from human immunodeficiency virus (HIV)-l-infected patients augment HIV infection mediated by Fe receptor uptake by human monocytes and the monocytic cell line U937. Antibody-dependent enhancement (ADE) and neutralization activity were studied in the sera of HIV -1 antibody-negative volunteers who had been immunized with three 40-llg doses of a recombinant gp 160 (rgp160) candidate HIV vaccine. Volunteers were vaccinated with rgp 160 or a hepatitis B vaccine as a control on days 0, 30, and 180. Sera were obtained before and after three doses of vaccine and were tested for ADE and neutralization activity. Serum samples collected before vaccination showed neither neutralization nor ADE activity. Thirteen sera from volunteers who received gp160 and four from placebo recipients failed to show ADE. Three sera showed low levelsof neutralization of strain III n ofHIV. Vaccination with this dose of rgp160 produced neutralizing antibodies in some subjects but did not induce detectable enhancing antibodies.
546
lID 1992; 165 (March)
Concise Communications
recipients of the recombinant gp 160 (rgp 160) HIV vaccine for ADE and neutralization.
Methods
B
A 35
30 LS
25
1 C
20
~
G. 15
10
·
m~~~
.,.....um
05
~ 11111 10·1
10·2
10·3
10·4
10·5
10·'
10·7
./0 MNm
10·1
10·2
SERUM DILUTION
10·3
10·4
10·5
10·'
10·1
1114
1/121
1/211
SERUM DILUTION
C
D
35
14
12
25
·
15
·
10
E
.. Q
c
~
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.,.....m
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10·2
10·3
10·4
10·5
10·'
10·1
./0 ,"",III
114
111
1/11
III 1132
SERUM DILUTION
SERUM DILUTION
Figure 1. Results of antibody-dependent enhancement experiments using different sera. Each group shows p24 from supernatant harvested on day 5 from U937 cells infected without serum (human immunodeficiency virus [HIV] alone) or U937 cells infected with HIV plus various dilutions of serum. Absolute p24 values varied between experiments because different lots of virus were used. A, Serum from HIV -infected donor. B, Serum from individual before immunization. C and D, Serum from volunteers after three doses of 40 Ilg of recombinant gp 160. D shows neutralization without enhancement.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 25, 2015
Vaccine trial. The rgpl60 was derived from a molecular clone of HIV (pNL4-3, similar to the III B strain). The gene was expressed in a baculovirus expression vector and the protein was expressed in insect cells (Microgenesys). The gp 160 was extracted, purified, and formulated with alum [13]. Healthy volunteers were determined to be HIV-uninfected by ELISA, Western blot, virus culture, and polymerase chain reaction. Subjects received 40 J,Lg of rgp 160 or 10 J,Lg of hepatitis B vaccine [13]. Subjects were immunized on days 0, 30, and 180 and blood was collected on days 0 and 200 for ADE studies. ADE studies. The ADE assay used U937 cells, a monocytic cell line that expresses CD4, FcR-I, and FcR-II. The methods of the ADE assay have been described in detail [10]. Serial dilu-
tions of serum from vaccine recipients or control HIV-positive patients were incubated with 0.1 ml ofHIV-l IIIB at 4°C for 30 min to form virus-antibody complexes. The virus-antibody complexes were used to infect 0.5 X 106 U937 cells at 3rC for I h. Cells were also infected with HIV incubated with medium alone. The MOl was 0.0 I. After infection, cells were washed twice and cultured in duplicate wells on 24-well plates. Medium was added on day 3, and supernatant was harvested on day 5 for p24 assay by ELISA (Du Pont, Wilmington, DE). The enhancement index (EI) was calculated as follows: EI = (p24 from cells infected with HIV-antibody complexesjp24 from cells infected with HIV alone). Neutralization was also assessed by determining the reduction of p24 from cells infected with low dilutions of antibody plus HIV compared with cells infected with HIV alone. The neutralizing index (NI) was calculated as follows: NI = (p24 from cells infected with virus alonejp24 from cells infected with virus and low dilution of serum).
JlD 1992;165 (March)
Concise Communications
Results
Table 1. Results of antibody-dependent enhancement experiments using sera from recipients of recombinant gp 160 or hepatitis B vaccine for human immunodeficiency virus determined by Western blot, enhancement index (EI), and neutralization index (NI). Western blot! Sample A B C
D E F G H 1 J K L M
N 0 p Q
Vaccine*
gpl60
gpl20
Gpl60 Gpl60 Gpl60 Gpl60 Gpl60 Gpl60 HepB Hep B Gpl60 Gpl60 Hep B Gpl60 Gpl60 Gpl60 Gpl60 Gpl60 HepB
+ + ++ +
++
+
+ +
±
+ +
gp41
EI
NI
+
±
±
+ ++
1.0 1.2 1.0 1.0 1.2 1.0 1.1 1.3 1.2 1.1 I.I 1.0 1.0 1.2 1.2 0.7 1.0
10.6 11.8 22.8 2.5 1.2 1.0 1.8 1.7 1.5 0.9 0.9 1.4 l.l 1.7 0.8 2.4 1.2
+ +
+
+ + +
±
±
+ ±
+ +
++
++
+
* Dose was 40 J.Lg of gp 160 or 10 J.Lg of hepatitis B (hep B) vaccine. t ++, strong positive; +, positive; ±, borderline positive; -, negative.
Three sera from volunteers immunized with 40 I-tg of rgp 160 showed neutralization. Figure 10 shows neutralization from one serum. The NI is 21, and the 50% neutralization titer (titer where p24 is reduced by 50%) is 1:32.
Discussion There was no evidence of HIV -1 ADE activity in the sera of these volunteers vaccinated with three doses of 40 I-tg of a baculovirus-derived rgp 160 candidate HIV-1 vaccine. This enhancement assay used heat-inactivated serum, which destroys complement, and measured ADE mediated by FcR. Previous work [10] has shown that the FcR part of IgG is necessary for enhancement. It is possible that HIV -1 could enter cells via two receptors (the CD4 receptor or the FcR of IgG) when virus is complexed with antibody. If HIV enters cells via the FcR, then antibodies to HIV might augment infection rather than neutralize it. ADE is observed when low concentrations of antibodies are present. In sera of HI VI-infected individuals, this usually occurs at dilutions of 10- 3 to 10-6 , beyond dilutions (10- 1-10- 2 ) where neutralization is observed. There is no evidence for an in vivo role of ADE in HIV-I infections, but theoretically, low vaccine doses, which might induce low levels of antibody, might be more likely to produce enhancement. The results of the present experiments, however, suggest that the gp 160 vaccine does not induce ADE. Low levels of neutralization were also found in 3 of 13 serum samples from recipients of three doses of vaccine. Dolin et al. [13] found no neutralization or complement-mediated enhancement until a fourth dose ofgp 160 was given I year after the third dose. They observed neutralization in 5 of 24 samples tested in an MT-2 cell assay. ADE mediated by complement receptors was found in 6 of24, and they did not measure uptake via Fe receptors [13]. Although the magnitude of enhancement demonstrated using sera from HIV -L-infected subjects is low (only two- to threefold increase in virus yield), it might be significant over the protracted course of HIV infection. The epitopes that induce enhancement rather than neutralization have not been elucidated. Using human monoclonal antibodies to HIV-I gp120, it has been recently demonstrated that distinct sites on gp 120 can induce a neutralizing monoclonal antibody (without any ADE activity) or an enhancing monoclonal antibody (without neutralizing activity) (unpublished data). Until enhancing epitopes have been mapped, we agree with Bolognesi [14] that it will be important to reduce that possibility of inducing enhancing bodies by vaccination.
References I. Desrosiers RC, Wyand MS, Kodama T, et al. Vaccine protection against simian immunodeficiency virus infection. Proc Nat! Acad Sci USA 1989;86:6353-7.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 25, 2015
Indices. Each experiment included a posiuve control serum from an HIV-I antibody-positive donor. The control was considered positive if p24 production from U937 cells infected with virus plus antibody was 2 SO above the p24 value from cells infected with virus alone. Control sera. The positive control sera used in each experiment were obtained from HIV antibody-positive donors. When U937 cells were infected with HIV-l IIIB plus low dilutions of such sera, the p24 yield was lower than when cells were infected with HIV alone. At higher sera dilutions, p24 production in U937 cells was increased compared with cells infected with virus alone (figure lA). Sera from vaccine recipients. Figure 1B shows a typical experiment using serum from a volunteer before vaccination. No enhancement or neutralization is seen. No day 0 sample from any vaccine recipient showed neutralization or enhancement activity (data not shown). The overall p24 values differed between experiments because different lots of HI V1IIIB virus were used in these assays. Sera from volunteers immunized with three doses of either 40 I-tg of rgp 160 or 10 I-tg of hepatitis B vaccine were tested for neutralization and ADE of HIV-1. Serum from vaccinated individuals failed to show evidence of ADE, while control sera run in the same experiment were positive for ADE (table 1). Figure lC shows a typical experiment using serum from a volunteer vaccinated with three doses of rgpl60.
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2. Murphey-Corb M, Martin LN, Davison-Fairburn B, et al. A formalin inactivated whole SIV vaccine confers protection in macaques. Science 1989;246:1293-7. 3. Carlson JR, McGraw TP, Keddie E, et al. Vaccine protection of rhesus macaques against simian immunodeficiency virus infection. AIDS Res Hum Retroviruses 1990;6: 1239-46. 4. Berman PW, Gregory TJ, Riddle L, et al. Protection of chimpanzees from infection by HIV -I after vaccination with recombinant glycoprotein gpl20 but not gp160. Nature 1990;345:622-5. 5. Hawkes RA. Enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls. Aust J Exp Med Sci 1964; 42:465-82. 6. Halstead SB, O'Rourke EJ. Dengue viruses and mononuclear phagecytes. Infection enhancement by nonneutralizing antibody. J Exp Med 1977; 146:20 1-17.
8. Cheng-Mayer C. Rutka JT, Rosenblum ML, et al. Human immunodeficiency virus can productively infect cultured human glial cells. Proc Natl Acad Sci USA 1987;84:3526-30. 9. Chehimi J, Bandyopadhyay S, Prakash K, et al. In vitro infection ofNK cells with different types of HIV type I isolates. J Virol 1991; 65: 1812-22. 10. Takeda A, Tuazon CU, Ennis FA. Antibody-enhanced infection by HIV-I via Fc receptor-mediated entry. Science 1988;242:580-3. II. Takeda A, Sweet RW, Ennis FA. Two receptors are required for antibody-dependent enhancement of human immunodeficiency virus type I infection: CD4 and FcR. J Virol 1990;64:5605-10. 12. Takeda A, Ennis FA. FcR mediated enhancement of HIV-I infection by antibody. AIDS Res Hum Retroviruses 1990;6:999-1004. 13. Dolin R, Graham BS, Greenberg SB, et al. The safety and immunogenicity of a human immunodeficiency virus type I (HIV -I) recombinant gp 160 candidate vaccine in humans. Ann Intern Med 1991;114:119-127. 14. Bolognesi DP. Do antibodies enhance infection ofcells by HIV? Nature 1989;340:431-2.
Failure of Prophylactic Ganciclovir to Prevent Cytomegalovirus Disease in Recipients of Lung Transplants Thomas C. Bailey, Elbert P. Trulock, Neil A. Ettinger, Gregory A. Storch, Joel D. Cooper, and William G. Powderly
Divisions of Infectious Diseases and Respiratory and Critical Care Medicine, Department of Medicine and Division of Thoracic Surgery, Department ofSurgery, Washington University School ofMedicine, St. Louis, Missouri
In an effort to prevent cytomegalovirus (CMV) pneumonitis, seven consecutive CMV-seronegative lung transplant recipients of organs from seropositive donors (D+/R-) were given ganciclovir, 2.5-5 mg/kg intravenously twice daily for the first 10-21 days after transplantation, and commercial polyvalent immune globulin, 200-400 mg/kg every 7-14 days intravenously, for the first 2-3 weeks after transplantation. This regimen was followed by oral acyclovir. Six patients developed CMV viremia and all developed CMV pneumonitis. Viremia occurred later in these patients compared with D+/R- patients who received alternative forms of CMV prophylaxis or CMV-seropositive recipients who received no specific prophylaxis (P = .023 and P = .021, respectively). There was no statistical difference in incidence or time to onset ofCMV pneumonitis. When given as described, prophylactic ganciclovir and immune globulin followed by oral acyclovir may have delayed CMV viremia but did not prevent it or pneumonitis in high-risk lung transplant recipients.
Cytomegalovirus (CMV) infection is an important and frequent complication of solid organ transplantation [I]. The CMV serologic status of the donor and the recipient are the
Received 18 July 1991; revised 25 October 1991. Presented in part: 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, 1991, Chicago (abstract 778). Financial support: Barnes HospitaL Reprints or correspondence: Dr. Thomas Bailey, Division of Infectious Diseases, Box 8051, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. The Journal of Infectious Diseases 1992;165:548-52 © 1992 by The University of Chicago. All rights reserved. 0022-\899/92/6503-0022$0 \.00
principal determinants of the risk ofinfection and for disease severity. Seronegative recipients transplanted with organs from seropositive donors (D+/R-) are at the highest risk for serious disease. The transplanted lung appears to be at particular risk of CMV pneumonitis, prompting some centers to match CMV-negative recipients with CMV-negative donors [2]. However, with a limited supply of donor organs, this approach is not always practical. In addition to the morbidity and mortality directly associated with CMV disease, the sequelae of this disease are potentially serious; they include an increased risk ofopportunistic infection, secondary bacterial infection, and transplant rejection [3-5]. Accordingly, there has been considerable in-
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 25, 2015
7. Dalgleish AG, Beverly PCL, Clapham PR, et al. The CD4 (T4) antigen is an essential component of the receptor of the AIDS retrovirus. Nature 1984;312:763-7.
JID 1992; 165 (March)
Studies of Antibody-Dependent Enhancement of Human Immunodeficiency Virus (HIV) Type 1 Infection Mediated by Fe Receptors Using Sera from Recipients of a Recombinant gp160 Experimental HIV-l Vaccine Richard H. Haubrich,* Akira Takeda, Wayne Koff, Gale Smith, and Francis A. Ennis
University of Massachusetts Medical Center, Worcester; AIDS Vaccine Evaluation Groups, National Institute for Allergy and Infectious Diseases. National Institutes of Health. Bethesda. Maryland; Microgenesys, Inc.. Meriden, Connecticut
Human immunodeficiency virus (HIV) infection is a tremendous global problem. During 1980-1990, > 161 ,000 cases of AIDS were reported in the United States. Intense efforts are underway to develop treatments for HIV and its resultant opportunistic infections. To prevent new HIV infections, a safe, effective vaccine is needed. Until recently, vaccine development was hindered by the inability to show protection from retroviral infections in animals. Three groups have recently reported protection against simian immunodeficiency virus challenge in monkeys [1-3]. A degree of protection against HIV-1 has also been reported in challenged chimpanzees [4]. One theoretical risk of vaccination is that antibodies to HIV might augment infection instead of neutralizing the virus. This augmentation ofinfection by antibodies is termed antibody-dependent enhancement (ADE). ADE was first described in animal viruses by Hawkes [5] and has been characterized most extensively for dengue virus. Studies on ADE in dengue have used convalescent-phase sera. Subneutralizing concentrations of antibody-augmented dengue virus yield 2-
Received 25 July 1991; revised 24 October 1991. Presented in part: VII International Conference on AIDS. June 1991, Florence. Italy (abstract MA 1327). All subjects gave informed consent. Financial support: National Institutes of Health (AI-24750. AI-26458. DAMD-C-279, AI-Onn, AI-25831). Reprints or correspondence: Dr. Francis A. Ennis, University of Massachusetts Medical Center, Division oflnfectious Diseases and Immunology, 55 Lake Ave. N., Worcester, MA 01655. * Present address: University of California San Diego Medical Center. The Journal of Infectious Diseases 1992;165:545-8 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6503-0021 $0 1.00
to 200-fold. The enhancing activity was in the IgG fraction of serum. Complement was not required for enhancement, but the Fe part ofIgG was necessary [6]. HIV is known to infect cells via the CD4 receptor [7]. T cells, rnonocytes, and monocytic cell lines are infected via this receptor. Several studies suggest that CD4 is not the only receptor for HIV. Glial cells, which do not have CD4, and NK cells, which have FcR-III (Fe receptor III ofIgG) but not CD4, have been infected with HIV [8, 9]. There are three possible mechanisms by which HIV could enter monocytic cells: nonspecific phagocytosis, CD4-mediated binding followed by fusion or endocytosis, and endocytosis of HI V-antibody complexes via the FcR ofIgG or the complement receptor. We have shown that HIV infection of the monocytic cell line U937 [10] and human monocytes [11] can be increased by adding subneutralizing concentrations of anti-HlV antibodies. This enhancing activity is present in the IgG fraction of serum and requires the FcR ofIgG and CD4 [11, 12]. The clinical significance of ADE has not been determined. In vitro experiments show that virus yield is increased two- to threefold in the presence oflow concentrations of antibody [10]. This difference is small compared with ADE with dengue in vitro. However, HIV infection lasts several years, and twofold increases in viral yield over thousands of infectious cycles in vivo may increase the viral burden and contribute to the pathogenesis of HI V infection. Although there are no in vivo data that demonstrate a role for ADE, it is considered a theoretical risk of HIV vaccination if the vaccine contains epitopes that induce enhancing antibodies. The epitopes that enhance infection have not been defined, and until the epitopes are mapped, it will be important to test vaccines for ADE. We tested sera from
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 25, 2015
Subneutralizing concentrations of sera from human immunodeficiency virus (HIV)-l-infected patients augment HIV infection mediated by Fe receptor uptake by human monocytes and the monocytic cell line U937. Antibody-dependent enhancement (ADE) and neutralization activity were studied in the sera of HIV -1 antibody-negative volunteers who had been immunized with three 40-llg doses of a recombinant gp 160 (rgp160) candidate HIV vaccine. Volunteers were vaccinated with rgp 160 or a hepatitis B vaccine as a control on days 0, 30, and 180. Sera were obtained before and after three doses of vaccine and were tested for ADE and neutralization activity. Serum samples collected before vaccination showed neither neutralization nor ADE activity. Thirteen sera from volunteers who received gp160 and four from placebo recipients failed to show ADE. Three sera showed low levelsof neutralization of strain III n ofHIV. Vaccination with this dose of rgp160 produced neutralizing antibodies in some subjects but did not induce detectable enhancing antibodies.
546
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Concise Communications
recipients of the recombinant gp 160 (rgp 160) HIV vaccine for ADE and neutralization.
Methods
B
A 35
30 LS
25
1 C
20
~
G. 15
10
·
m~~~
.,.....um
05
~ 11111 10·1
10·2
10·3
10·4
10·5
10·'
10·7
./0 MNm
10·1
10·2
SERUM DILUTION
10·3
10·4
10·5
10·'
10·1
1114
1/121
1/211
SERUM DILUTION
C
D
35
14
12
25
·
15
·
10
E
.. Q
c
~
· OS
.,.....m
10·1
10·2
10·3
10·4
10·5
10·'
10·1
./0 ,"",III
114
111
1/11
III 1132
SERUM DILUTION
SERUM DILUTION
Figure 1. Results of antibody-dependent enhancement experiments using different sera. Each group shows p24 from supernatant harvested on day 5 from U937 cells infected without serum (human immunodeficiency virus [HIV] alone) or U937 cells infected with HIV plus various dilutions of serum. Absolute p24 values varied between experiments because different lots of virus were used. A, Serum from HIV -infected donor. B, Serum from individual before immunization. C and D, Serum from volunteers after three doses of 40 Ilg of recombinant gp 160. D shows neutralization without enhancement.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 25, 2015
Vaccine trial. The rgpl60 was derived from a molecular clone of HIV (pNL4-3, similar to the III B strain). The gene was expressed in a baculovirus expression vector and the protein was expressed in insect cells (Microgenesys). The gp 160 was extracted, purified, and formulated with alum [13]. Healthy volunteers were determined to be HIV-uninfected by ELISA, Western blot, virus culture, and polymerase chain reaction. Subjects received 40 J,Lg of rgp 160 or 10 J,Lg of hepatitis B vaccine [13]. Subjects were immunized on days 0, 30, and 180 and blood was collected on days 0 and 200 for ADE studies. ADE studies. The ADE assay used U937 cells, a monocytic cell line that expresses CD4, FcR-I, and FcR-II. The methods of the ADE assay have been described in detail [10]. Serial dilu-
tions of serum from vaccine recipients or control HIV-positive patients were incubated with 0.1 ml ofHIV-l IIIB at 4°C for 30 min to form virus-antibody complexes. The virus-antibody complexes were used to infect 0.5 X 106 U937 cells at 3rC for I h. Cells were also infected with HIV incubated with medium alone. The MOl was 0.0 I. After infection, cells were washed twice and cultured in duplicate wells on 24-well plates. Medium was added on day 3, and supernatant was harvested on day 5 for p24 assay by ELISA (Du Pont, Wilmington, DE). The enhancement index (EI) was calculated as follows: EI = (p24 from cells infected with HIV-antibody complexesjp24 from cells infected with HIV alone). Neutralization was also assessed by determining the reduction of p24 from cells infected with low dilutions of antibody plus HIV compared with cells infected with HIV alone. The neutralizing index (NI) was calculated as follows: NI = (p24 from cells infected with virus alonejp24 from cells infected with virus and low dilution of serum).
JlD 1992;165 (March)
Concise Communications
Results
Table 1. Results of antibody-dependent enhancement experiments using sera from recipients of recombinant gp 160 or hepatitis B vaccine for human immunodeficiency virus determined by Western blot, enhancement index (EI), and neutralization index (NI). Western blot! Sample A B C
D E F G H 1 J K L M
N 0 p Q
Vaccine*
gpl60
gpl20
Gpl60 Gpl60 Gpl60 Gpl60 Gpl60 Gpl60 HepB Hep B Gpl60 Gpl60 Hep B Gpl60 Gpl60 Gpl60 Gpl60 Gpl60 HepB
+ + ++ +
++
+
+ +
±
+ +
gp41
EI
NI
+
±
±
+ ++
1.0 1.2 1.0 1.0 1.2 1.0 1.1 1.3 1.2 1.1 I.I 1.0 1.0 1.2 1.2 0.7 1.0
10.6 11.8 22.8 2.5 1.2 1.0 1.8 1.7 1.5 0.9 0.9 1.4 l.l 1.7 0.8 2.4 1.2
+ +
+
+ + +
±
±
+ ±
+ +
++
++
+
* Dose was 40 J.Lg of gp 160 or 10 J.Lg of hepatitis B (hep B) vaccine. t ++, strong positive; +, positive; ±, borderline positive; -, negative.
Three sera from volunteers immunized with 40 I-tg of rgp 160 showed neutralization. Figure 10 shows neutralization from one serum. The NI is 21, and the 50% neutralization titer (titer where p24 is reduced by 50%) is 1:32.
Discussion There was no evidence of HIV -1 ADE activity in the sera of these volunteers vaccinated with three doses of 40 I-tg of a baculovirus-derived rgp 160 candidate HIV-1 vaccine. This enhancement assay used heat-inactivated serum, which destroys complement, and measured ADE mediated by FcR. Previous work [10] has shown that the FcR part of IgG is necessary for enhancement. It is possible that HIV -1 could enter cells via two receptors (the CD4 receptor or the FcR of IgG) when virus is complexed with antibody. If HIV enters cells via the FcR, then antibodies to HIV might augment infection rather than neutralize it. ADE is observed when low concentrations of antibodies are present. In sera of HI VI-infected individuals, this usually occurs at dilutions of 10- 3 to 10-6 , beyond dilutions (10- 1-10- 2 ) where neutralization is observed. There is no evidence for an in vivo role of ADE in HIV-I infections, but theoretically, low vaccine doses, which might induce low levels of antibody, might be more likely to produce enhancement. The results of the present experiments, however, suggest that the gp 160 vaccine does not induce ADE. Low levels of neutralization were also found in 3 of 13 serum samples from recipients of three doses of vaccine. Dolin et al. [13] found no neutralization or complement-mediated enhancement until a fourth dose ofgp 160 was given I year after the third dose. They observed neutralization in 5 of 24 samples tested in an MT-2 cell assay. ADE mediated by complement receptors was found in 6 of24, and they did not measure uptake via Fe receptors [13]. Although the magnitude of enhancement demonstrated using sera from HIV -L-infected subjects is low (only two- to threefold increase in virus yield), it might be significant over the protracted course of HIV infection. The epitopes that induce enhancement rather than neutralization have not been elucidated. Using human monoclonal antibodies to HIV-I gp120, it has been recently demonstrated that distinct sites on gp 120 can induce a neutralizing monoclonal antibody (without any ADE activity) or an enhancing monoclonal antibody (without neutralizing activity) (unpublished data). Until enhancing epitopes have been mapped, we agree with Bolognesi [14] that it will be important to reduce that possibility of inducing enhancing bodies by vaccination.
References I. Desrosiers RC, Wyand MS, Kodama T, et al. Vaccine protection against simian immunodeficiency virus infection. Proc Nat! Acad Sci USA 1989;86:6353-7.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 25, 2015
Indices. Each experiment included a posiuve control serum from an HIV-I antibody-positive donor. The control was considered positive if p24 production from U937 cells infected with virus plus antibody was 2 SO above the p24 value from cells infected with virus alone. Control sera. The positive control sera used in each experiment were obtained from HIV antibody-positive donors. When U937 cells were infected with HIV-l IIIB plus low dilutions of such sera, the p24 yield was lower than when cells were infected with HIV alone. At higher sera dilutions, p24 production in U937 cells was increased compared with cells infected with virus alone (figure lA). Sera from vaccine recipients. Figure 1B shows a typical experiment using serum from a volunteer before vaccination. No enhancement or neutralization is seen. No day 0 sample from any vaccine recipient showed neutralization or enhancement activity (data not shown). The overall p24 values differed between experiments because different lots of HI V1IIIB virus were used in these assays. Sera from volunteers immunized with three doses of either 40 I-tg of rgp 160 or 10 I-tg of hepatitis B vaccine were tested for neutralization and ADE of HIV-1. Serum from vaccinated individuals failed to show evidence of ADE, while control sera run in the same experiment were positive for ADE (table 1). Figure lC shows a typical experiment using serum from a volunteer vaccinated with three doses of rgpl60.
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2. Murphey-Corb M, Martin LN, Davison-Fairburn B, et al. A formalin inactivated whole SIV vaccine confers protection in macaques. Science 1989;246:1293-7. 3. Carlson JR, McGraw TP, Keddie E, et al. Vaccine protection of rhesus macaques against simian immunodeficiency virus infection. AIDS Res Hum Retroviruses 1990;6: 1239-46. 4. Berman PW, Gregory TJ, Riddle L, et al. Protection of chimpanzees from infection by HIV -I after vaccination with recombinant glycoprotein gpl20 but not gp160. Nature 1990;345:622-5. 5. Hawkes RA. Enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls. Aust J Exp Med Sci 1964; 42:465-82. 6. Halstead SB, O'Rourke EJ. Dengue viruses and mononuclear phagecytes. Infection enhancement by nonneutralizing antibody. J Exp Med 1977; 146:20 1-17.
8. Cheng-Mayer C. Rutka JT, Rosenblum ML, et al. Human immunodeficiency virus can productively infect cultured human glial cells. Proc Natl Acad Sci USA 1987;84:3526-30. 9. Chehimi J, Bandyopadhyay S, Prakash K, et al. In vitro infection ofNK cells with different types of HIV type I isolates. J Virol 1991; 65: 1812-22. 10. Takeda A, Tuazon CU, Ennis FA. Antibody-enhanced infection by HIV-I via Fc receptor-mediated entry. Science 1988;242:580-3. II. Takeda A, Sweet RW, Ennis FA. Two receptors are required for antibody-dependent enhancement of human immunodeficiency virus type I infection: CD4 and FcR. J Virol 1990;64:5605-10. 12. Takeda A, Ennis FA. FcR mediated enhancement of HIV-I infection by antibody. AIDS Res Hum Retroviruses 1990;6:999-1004. 13. Dolin R, Graham BS, Greenberg SB, et al. The safety and immunogenicity of a human immunodeficiency virus type I (HIV -I) recombinant gp 160 candidate vaccine in humans. Ann Intern Med 1991;114:119-127. 14. Bolognesi DP. Do antibodies enhance infection ofcells by HIV? Nature 1989;340:431-2.
Failure of Prophylactic Ganciclovir to Prevent Cytomegalovirus Disease in Recipients of Lung Transplants Thomas C. Bailey, Elbert P. Trulock, Neil A. Ettinger, Gregory A. Storch, Joel D. Cooper, and William G. Powderly
Divisions of Infectious Diseases and Respiratory and Critical Care Medicine, Department of Medicine and Division of Thoracic Surgery, Department ofSurgery, Washington University School ofMedicine, St. Louis, Missouri
In an effort to prevent cytomegalovirus (CMV) pneumonitis, seven consecutive CMV-seronegative lung transplant recipients of organs from seropositive donors (D+/R-) were given ganciclovir, 2.5-5 mg/kg intravenously twice daily for the first 10-21 days after transplantation, and commercial polyvalent immune globulin, 200-400 mg/kg every 7-14 days intravenously, for the first 2-3 weeks after transplantation. This regimen was followed by oral acyclovir. Six patients developed CMV viremia and all developed CMV pneumonitis. Viremia occurred later in these patients compared with D+/R- patients who received alternative forms of CMV prophylaxis or CMV-seropositive recipients who received no specific prophylaxis (P = .023 and P = .021, respectively). There was no statistical difference in incidence or time to onset ofCMV pneumonitis. When given as described, prophylactic ganciclovir and immune globulin followed by oral acyclovir may have delayed CMV viremia but did not prevent it or pneumonitis in high-risk lung transplant recipients.
Cytomegalovirus (CMV) infection is an important and frequent complication of solid organ transplantation [I]. The CMV serologic status of the donor and the recipient are the
Received 18 July 1991; revised 25 October 1991. Presented in part: 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, 1991, Chicago (abstract 778). Financial support: Barnes HospitaL Reprints or correspondence: Dr. Thomas Bailey, Division of Infectious Diseases, Box 8051, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. The Journal of Infectious Diseases 1992;165:548-52 © 1992 by The University of Chicago. All rights reserved. 0022-\899/92/6503-0022$0 \.00
principal determinants of the risk ofinfection and for disease severity. Seronegative recipients transplanted with organs from seropositive donors (D+/R-) are at the highest risk for serious disease. The transplanted lung appears to be at particular risk of CMV pneumonitis, prompting some centers to match CMV-negative recipients with CMV-negative donors [2]. However, with a limited supply of donor organs, this approach is not always practical. In addition to the morbidity and mortality directly associated with CMV disease, the sequelae of this disease are potentially serious; they include an increased risk ofopportunistic infection, secondary bacterial infection, and transplant rejection [3-5]. Accordingly, there has been considerable in-
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