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Mechanisms of human immunodeficiency virus Type 1 (HIV-1) neutralization: irreversible inactivation of infectivity by anti-HIV-1 antibody.

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Copyrightq1996, American Society for Microbiology

Mechanisms of Human Immunodeficiency Virus Type 1

(HIV-1) Neutralization: Irreversible Inactivation of

Infectivity by Anti-HIV-1 Antibody

J. STEVEN MCDOUGAL,* M. SUSAN KENNEDY, SHERRY L. ORLOFF,

JANET K. A. NICHOLSON,ANDTHOMAS J. SPIRA

Immunology Branch, Division of HIV/AIDS, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta Georgia 30333

Received 26 September 1995/Accepted 25 April 1996

An assay for the neutralization of human immunodeficiency virus type 1 (HIV-1) is described in which the reduction in infectious titer of HIV-1 after preincubation at 37&C with antibody-positive serum is the measure of neutralization. The assay format and its controls allow several experimental manipulations that, taken together, indicate an effect of antibody on HIV-1 infectivity that occurs before or independently of HIV-1 attachment. The direct inactivation of HIV-1 infectivity by antibody is irreversible and temperature dependent, requires a bivalent antibody directed against accessible envelope determinants, and does not require a heat-labile or Ca21- or Mg21-dependent cofactor. The mechanism of inactivation cannot be explained by

agglutination of virus, nor is it associated with disruption or dissociation of envelope protein from virions. Rather, the antibody is likely to perturb some metastable property of the envelope that is required for entry. Laboratory-adapted HIV-1 isolates were more sensitive to the inactivating effects of sera than were primary patient isolates. The latter were particularly resistant to inactivation by contemporary autologous sera, a feature not explained by blocking antibodies. Additional studies showed a weak relationship between disease course and serum inactivation of the reference LAI laboratory strain of HIV-1. Heteroduplex analysis and autologous inactivation assays of sequential specimens from individual patients indicate that over time, the viral quasispecies that emerge and dominate are resistant to the inactivating effects of earlier sera.

In vitro, antibody to human immunodeficiency virus type 1 (HIV-1) can prevent or neutralize infection, and antibody has been shown to afford protective immunity in vivo in chimpan-zees (14, 15, 51). HIV-1-infected people mount and sustain a vigorous antibody response to HIV-1. A portion of this re-sponse is neutralizing. The presence or quantity of neutralizing antibody, as measured by existing in vitro neutralization assays, has been associated with the healthy carrier state, but the relationship to control of disease progression or severity re-mains controversial (reviewed in reference 26). Despite the presence of neutralizing antibodies, infection persists and most patients ultimately succumb to AIDS. Persistent infection may reflect a dynamic interplay between immune recognition of HIV-1 species and the emergence of viral populations that escape recognition (1, 2, 10, 35, 42, 62).

Many studies have examined the neutralization response in HIV-1 infection. Most of these studies have focused on defin-ing determinants that are targets for neutralizdefin-ing antibodies (65), virtually all of which reside on env-encoded proteins gp120 and gp41 (38). Relatively little attention has been paid to the mechanism by which antibody neutralizes virus. One exception is the analysis of inhibition of HIV-1 infection by soluble CD4 (sCD4). sCD4 could be considered analogous to a class of antibodies that are reactive with conformation-de-pendent determinants and that block receptor binding. Two mechanisms, receptor blockade and direct inactivation of HIV-1 infectivity, have been described (25, 39, 40, 47). While it may seem most plausible that antibody blocks infection by interfering with attachment to target cells, antibody reaction

with some neutralizing determinants, namely, the V3 region of gp120, does not block binding yet effectively inhibits infection (60). Effects of antibody independent of receptor engagement (pre- or postattachment effects) have been described in other viral systems where, in some cases, they appear to be the prominent and important mechanisms (4, 11, 18, 22, 69). This report describes a modified neutralization assay that reveals a mechanism of antibody inhibition that can occur prior to and independently of HIV-1 attachment.

MATERIALS AND METHODS

Virus stocks and cell lines.The LAI, RF, MN, and JR-CSF strains of HIV-1 were propagated in phytohemagglutinin (PHA)-stimulated normal peripheral blood lymphocytes (PBL) (32). The LAI strain was obtained from J.-C. Cher-mann (Institut Pasteur, Paris, France); RF and MN were obtained from the AIDS Research and Reference Repository Program (ARRRP), Bethesda, Md.; and JR-CSF was obtained from I. S. Y. Chen (University of California, Los Angeles). Primary isolates were obtained by coculture of lymphocytes from HIV-1-seropositive men with PHA-stimulated PBL in medium containing inter-leukin 2 (32, 47). They were passed one more time in PHA-stimulated PBL. CD41T-cell line C8166 was obtained from Robin Weiss (Chester Beatty Lab-oratories, London, England). HIV-IIIB-infected H9 cells and the uninfected parental cell line HT were obtained from R. C. Gallo (National Cancer Institute, Bethesda, Md.).

Virus neutralization and inactivation assays.Two assays for serum inhibition of HIV-1 infectivity, which we refer to as the neutralization and inactivation assays, were used (47). In the neutralization assay, an intended dose of 100 50% tissue culture-infective doses of HIV-1 (the actual input doses, determined in the same run, ranged from 50 to 700 tissue culture-infective doses) was mixed with graded doses of heat-inactivated (568C for 30 min) serum and incubated for 15 min at room temperature. The mixtures were added to PHA-stimulated PBL and incubated at 378C overnight. Washed cells were plated in microculture (10 cultures per dilution; 105

cells per culture) and monitored for virus production 8 and 12 days later by an antigen capture assay (30).

In the inactivation assay, 50ml of an undiluted HIV-1 stock was mixed 1:1 with heat-inactivated serum (final serum concentration, 1:4 unless otherwise stated) and incubated for 2 h at 378C. The titer of the mixture was then determined as described in detail elsewhere (30, 47). Briefly, after overnight incubation with

* Corresponding author. Mailing address: Bldg. 1-1202 (A25), Cen-ters for Disease Control, Atlanta GA 30333. Phone: (404) 639-3434. Fax: (404) 639-2108.

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serial 10-fold dilutions of the mixture, washed cells were dispensed into micro-culture (10 micro-cultures per dilution, 105PHA-stimulated PBL per culture). Eight and 12 days later, supernatants were monitored for viral replication by an antigen capture assay (30). Cultures were scored positive if the optical density at 490 nm was 3 standard deviations above the mean of 10 uninfected cultures run in the same plate. Two control titrations were performed for each serum. First, the serum–HIV-1 mixture was preincubated at 08C for 2 h instead of at 378C. Second, HIV-1 and serum were preincubated in separate tubes at 378C, dilutions were made, and the dilutions were combined just before addition to cells. Thus, the experimental and control cultures had identical amounts of HIV-1 and serum but differed in the opportunity for interaction at 378C before exposure to cells (47).

IgG purification and proteolytic digestion.Immunoglobulin G (IgG) was purified from heat-inactivated (568C for 30 min) anti-HIV human serum by two precipitations in 40% saturated ammonium sulfate followed by DEAE anion-exchange chromatography (16). F(ab9)2fragments were prepared by pepsin diges-tion as modified by Nisinoff for human IgG, followed by Sephadex G150 chroma-tography (44). Fab and Fc fragments were prepared by papain digestion, followed by anion- and cation-exchange chromatography as described by Franklin (17).

Envelope release from virions and radioimmunoprecipitation.An HIV-1LAI preparation was pelleted by centrifugation (145,0003g for 30 min) and

resus-pended to one-eighth of the original volume in phosphate-buffered saline (pH 7.2) containing 0.5% bovine serum albumin. A portion of the preparation was converted to a viral lysate by addition of Nonidet P-40 (0.75%, vol/vol) and heating at 568C for 30 min. Virus and viral lysate were incubated in buffer, a 1:10 final dilution of anti-gp120 monoclonal antibody (MAb) 9305 (Du Pont Corp., Wilmington, Del.), or 50mg of sCD4 (Progenics Pharmaceuticals, Tarrytown, N.Y.) per ml at 0 or 378C for 2 h in a total volume of 200ml. Virus particles were pelleted by centrifugation at 145,0003g for 30 min, and supernatant gp120 was

measured by enzyme-linked immunosorbent assay (33) with recombinant gp120 as the standard (Microgenesis, West Haven, Conn.). Anti-gp120 MAb 9305 does not interfere with gp120 detection in this assay, as assessed by testing of virus preparations in the presence or absence of the MAb.

An extracellular HIV-1LAIpreparation internally labelled with [35 S]methi-onine-cysteine was prepared as previously described (47) and processed by the procedure described above, except that pellet and supernatant materials were dissolved in detergent buffer and absorbed on immunoadsorbent beads coated with polyclonal human anti-HIV IgG (47). Absorbed material was eluted with sodium dodecyl sulfate sample buffer at 658C and analyzed by polyacrylamide gel electrophoresis (47).

Heteroduplex mobility assay.The heteroduplex mobility assay was performed as described by Delwart et al. (8). DNA was obtained from PBL of infected men, and primary HIV-1 isolate DNA was obtained from normal PHA-stimulated PBL that had been infected with HIV-1 isolates for 3 days. The preparation of DNA, by using a lysis buffer containing detergent and proteinase K (46), and the conditions for nested PCR have already been described (8, 46). We used first-round primers ED3 and ED14 and second-first-round primers ES7 and ES8 as described by Delwart et al. (8). These are expected to generate an envelope-encoded PCR fragment of approximately 700 bp (positions 6579 to 7245 [8]). The PCR products were purified by using a Qiagen PCR purification spin kit (Qiagen Inc., Chatsworth, Calif.). To ensure reasonable sampling of HIV-1 sequences in each preparation, the amount of starting DNA was at least 50-fold in excess of that required to generate a PCR product. Isolate DNA was internally radiolabelled with [32

P]dCTP in the second round of PCR amplification. Unla-belled PCR product DNA was mixed with radiolaUnla-belled DNA (estimated ratio, 80:1 to 160:1), heated at 948C for 2 min, and cooled on ice. Duplex formation was resolved on neutral, nondissociating 5% acrylamide gels (8) at 50 mA for 90 min at room temperature. Radiolabelled and unlabelled DNA contents of the mix-tures were determined by pretitration as follows. Enough radiolabelled DNA was used to display an ample signal after overnight development of the radioauto-graph but not so much that it formed homoduplexes when run by itself. Sufficient unlabelled DNA was used to form duplexes with the radiolabelled DNA and minimize the radiolabelled single-stranded band but not so much that excessive partially hybridized “tangles” were retained in the loading well of the gel.

Other assays.The following methods have been described by us in detail elsewhere: flow cytometric analysis of lymphocyte phenotypes from infected patients (43), flow cytometric analysis of binding to viral proteins on infected H9 cells (32), quantitation of infectious virus titers in separated lymphocytes from infected people (43), Western blot (immunoblot) determination of titers of band-specific antibodies to viral proteins (31), and immunofluorescence detec-tion of cytoplasmic viral antigens in acid-alcohol-fixed H9 cells (30). HIV-1 p24 core antigen levels were measured with a commercial kit (Abbott Laboratories, Chicago, Ill.).

RESULTS

Inactivation.An HIV inoculum (LAI strain) was mixed with human anti-HIV serum (1:4 final concentration) and incubated

for 2 h at 378C. Serial 10-fold dilutions were made and added

to cells; viral replication was monitored to determine the 50% infective dose. A representative assay is shown in Fig. 1. Two

control titrations were performed. In one, HIV and anti-HIV

serum were preincubated at 08C instead of 378C. In the other,

HIV and anti-HIV serum were preincubated at 378C in

sepa-rate tubes. Sepasepa-rate dilutions were made and combined just before addition to cells. Thus, the inactivation and control cultures contained identical amounts of HIV and anti-HIV

serum but differed in the opportunity for interaction at 378C

before addition to cells. A reduction in titer occurred under these conditions (Fig. 1). We attribute this reduction to an

inactivation of HIV that occurs at 378C. Furthermore, the

inac-tivation at 378C was not reversed by returning the antibody-virus

mixture to 08C for 2 h before dilution (data not shown).

The 2- to 3-log reduction in titer is significant. The mean

difference (6 the standard deviation) between log titers of

duplicate titrations in the same run (intrarun variation) was

0.2260.24 (n547 paired determinations). The mean interrun

variation in log titer was 0.5860.44 (n5109 pairs). The mean

difference between runs in reduction of log titer by sera was

0.3260.32 (n569 pairs).

A 2-h preincubation of serum with HIV at 378C resulted in

maximum inactivation for most sera (Fig. 2a), and the inacti-vating activity required relatively high concentrations of serum in the initial incubation (Fig. 2b). Inactivation was much slower

or did not occur at 08C. Neither extending the 08C

preincuba-tion for 6 h nor storing the serial 10-fold dilupreincuba-tions of samples

preincubated at 08C at 378C for 6 or 18 h before addition to

cells resulted in increased inactivation (data not shown). An occasional serum (one in five), however, did register

approxi-mately a 1-log lower titer in the 08C control titration than the

titer of the other two controls (separate dilution control or virus titration in the absence of antibody) but never as low as

that of the sample preincubated at 378C (see Discussion).

Thus, 378C incubation in the presence of a high concentration

of serum is the requisite for maximum inactivation in this system.

Inactivation is mediated by bivalent IgG anti-HIV antibod-ies and does not require a Ca21- or Mg21-dependent or

heat-labile cofactor. The IgG fraction from an anti-HIV human serum was purified and used to prepare the enzymatic

frag-ments F(ab9)2and Fab. When tested in equibinding amounts

[1:1:2 molar ratio of IgG to F(ab9)2to Fab] and at

concentra-FIG. 1. Inactivation of HIV-1 by anti-HIV-1 serum. An undiluted HIV-1LAI stock was mixed with anti-HIV-1 serum 5111 (1:4 final serum dilution) and preincubated for 2 h at 37 or 08C. The mixtures were diluted as indicated and added to cells in microculture, and virus replication was monitored. Symbols:F, 378C preincubation;■, 08C preincubation;å, separate preincubation of serum and virus at 378C, dilution, and combination of dilutions at addition to cell culture;E, virus titration without serum.

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tions similar to that in a 1:4 dilution of human serum, the

bivalent IgG and F(ab9)2 preparations inactivated HIV,

whereas the monovalent Fab preparation did not (Table 1). When heat-inactivated anti-HIV serum was tested in the usual amount (1:4) or at a lower concentration (1:10), supplementing the reaction with fresh human serum or EDTA-treated plasma did not augment or diminish activity (Table 1).

Inactivation by monospecific antibodies.A set of monospe-cific antibodies and antisera, all reportedly reactive with the test strain of HIV-1 (LAI), were tested in this assay (Table 2). These were selected from a larger panel, and we required that they be reactive in several in-house assays. All of the MAbs reacted with cytoplasmic HIV-1 antigens by indirect immuno-fluorescence with acid-alcohol-fixed, HIV-1-infected H9 cells

at a 1:100 dilution. In addition, all of the anti-gp120 monoclo-nal and polyclomonoclo-nal reagents were tested by immunoprecipita-tion of internally radiolabelled, detergent-lysed extracellular HIV-1, and all but one were reactive with gp120 (Table 2). Finally, all of the MAb reagents were tested for binding to the cell surface of HIV-1-infected H9 cells by indirect immunoflu-orescence and flow cytometry.

Neither of the two anti-gp41 reagents inactivated HIV-1. All four polyclonal anti-gp120 sera inactivated HIV-1. This in-cluded a set of three goat antisera reactive with the carboxyl half of gp120 that inactivated HIV-1 regardless of whether reactivity included (PB1 and PB1sub2) or excluded (PB1sub7) the V3 region of gp120. All of the V3-specific MAbs inacti-vated HIV-1. Two MAbs reactive with carboxyl-terminal epitopes of gp120 were inactive. The two conformation-depen-dent MAbs reactive with the CD4-binding domain (CD4bd) of gp120 inactivated the virus but were not as potent as the V3 MAbs or the polyclonal sera. These human MAbs were not as high titered as some of the other reagents but were tested in concentrations that were saturating in the assay for an H9 cell surface reaction.

The one feature that appears to predict whether a given anti-gp120 reagent will inactivate is reactivity with cell surface gp120. All that were positive in this assay were inactivating. The two anti-gp41 reagents did react with the cell surface envelope but did not inactivate the virus. In this case, the cell surface reaction may be with gp41 that is not associated with gp120 or from which gp120 has spontaneously been shed (56).

Mechanism of inactivation.One possible explanation for the apparent inactivation is that antibody agglutinates virus, thereby reducing the number of functional infectious units without actually reducing the number of infectious virions. To address this, we designed experiments to separately agglutinate

and inactivate HIV-1. An HIV-1LAIsuspension was incubated

with anti-HIV serum at 08C for 2 h, the preparation was

cen-trifuged at 6003 g for 20 min, and the supernatant was

re-moved. The pellet, resuspended in medium and defined for this purpose as agglutinated virus, was then further incubated

at 0 or 378C for 2 h. The suspensions were serially diluted, and

infectious titers were determined. Despite identical amounts of

initially agglutinated virus, the additional incubation at 378C

was required to reduce the titer (Fig. 3). Furthermore, optimal serum dilutions for maximum precipitation (agglutination) and for inactivation were different. Inactivation occurred at rela-tively high serum concentrations (Fig. 3b), whereas optimal precipitation occurred with lower serum concentrations (Fig. 3a). It is noteworthy that the sum of the infectious virus

con-tent in the 08C immunoprecipitate plus the content in its

su-pernatant was about 1 log less than the content of the starting

virus preparation or of the mixture kept at 08C and not

sepa-rated by centrifugation. (Titers in both the immunoprecipitate and the supernatant were further reduced or abolished by a

378C incubation before dilution.) Thus, some titer reduction

can occur at 08C, and physical packing of virions by

centrifu-gation may be required to demonstrate it optimally.

We took advantage of the fact that viral titers could be

recovered in virus-antibody immunoprecipitates formed at 08C

to address the question of reversibility. Virus-antibody

immu-noprecipitates were formed at 08C by using a 1:8 final dilution

of serum 5111 and HIV-1LAIand washed as described above.

The immunoprecipitates were incubated at 37 or 08C for 2 h,

and serial 10-fold dilutions were made. Instead of being

di-rectly added to cells, all dilutions were kept at 378C for 18 h to

allow elution of bound antibody from virus. A virus titer of

103.44was recovered in the immunoprecipitate preincubated at

08C, whereas no viral replication was detected in the

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immuno-FIG. 2. Preincubation time and serum concentrations required for HIV-1 inactivation. (a) HIV-1LAIand anti-HIV-1 sera (F, 5111;■, 5074;å, 5028) were mixed (1:4 final serum dilution) and preincubated for the indicated times at 378C, diluted, and added to cell culture, and the virus titer was determined. The zero time point is the titer of the mixture kept at 08C for 2 h. (b) HIV-1 was mixed with different final dilutions of sera and preincubated for 2 h at 378C, and viral titrations were performed. The zero dilution points represent virus titers without serum (the dilution control and 08C preincubation controls registered similar titers).

TABLE 1. Inactivation of HIV-1 by serum components or fractions

Expt and test material(s)

Log titer reduction

Expt 1 Expt 2

IgG fragmentsa

Anti-HIV IgG 3.13 3.72

Anti-HIV F(ab9)2 NDb 2.55

Anti-HIV Fab 0.80 0.60

Heat-labile or Ca21-dependent cofactorc

Anti-HIV serum .3.05d 2.99

Anti-HIV serum1NHS .3.05 3.03

Anti-HIV serum1heat-inactivated NHS .3.05 3.56

Anti-HIV serum1EDTA-NHS .3.05 2.56

aIgG, F(ab9)

2, and Fab fragments purified from serum 5111 were tested against HIVLAIat equibinding final concentrations (17.8, 19.7, and 36.0mM, respectively), similar to the concentration of IgG (16.6mM), in a 1:4 dilution of serum.

b

ND, not done.

c

HIVLAIwas incubated with a 1:4 (experiment 1) or 1:10 (experiment 2) final concentration of anti-HIV serum 5111 plus a 1:4 final concentration (in both experiments) of fresh NHS, heat-inactivated (568C for 30 min) NHS, or plasma from the same bleeding collected in EDTA.

d

The symbol.indicates that the experimental titer was,1.0 and, therefore, that the log reduction in titer was greater than the difference between the experimental and control titers.

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precipitate preincubated at 378C (titer, ,101.00). Similar

re-sults were obtained when the dilutions were kept at 08C rather

than at 378C for 18 h. Thus, conditions that allow removal of

excess antibody, inactivation at 378C, and dilution to allow

dissociation of bound antibody did not reverse the inactivation. With sCD4, inactivation of HIV-1 is associated with the induction of shedding of gp120 from virions, although it is not clear that the shedding phenomenon is a cause or concomitant of inactivation (39, 40). In one model of induced shedding, HIV-1-infected cells, after incubation with sCD4, lose cell sur-face expression of gp120 (56). We incubated HIV-1-infected H9 cells in medium with saturating concentrations of the

in-activating MAbs listed in Table 2 for 2 h at 37 or 08C and then

stained the cells with the appropriate fluorescein–anti-Ig

con-jugate at 08C. The fluorescence intensities (i.e., levels of

enve-lope expression) were essentially identical (620%) at both

temperatures. Under the same conditions, addition of sCD4, alone or with the MAbs, resulted in a reduction of fluorescence

intensity of 40 to 80% at 378C. The same experiments were

performed with polyclonal human sera, and as with the MAbs, no reduction in binding by human antibodies was found. How-ever, sCD4 (the positive control), when added with polyclonal sera, did not result in decreased staining as expected. Either polyclonal sera block the effect of sCD4 or sCD4 does induce gp120 shedding but the loss of gp120 staining is compensated for by increased staining of other viral structures (e.g., gp41) (56). It is possible that a variation of the latter process oc-curred with the gp120 MAbs as well. In this case, however, loss of gp120 would have had to be compensated for by increased epitope exposure on the remaining gp120 molecules.

To better explore the possibility that interaction of virions

with inactivating antibody at 378C results in disruption of

viri-ons (release of gp120), we incubated virus with an inactivating

anti-gp120 MAb at 0 and 378C for 2 h. The preparations were

ultracentrifuged to pellet virus particles, and gp120 content in the supernatant was measured. Under conditions in which

sCD4 induced substantial release of gp120, the MAb did not (Table 3). The use of saturating amounts of monospecific MAbs eliminated the potential problem of precipitating free gp120-MAb complexes during centrifugation to remove parti-cle-associated gp120. This is evidenced by the fact that virus lysates run in parallel registered similar supernatant envelope contents with and without added MAb. Two other inactivating MAbs were also tested in this assay with similar results. (Poly-clonal sera could not be used in this assay because they inter-fere with the immunologic detection of gp120.) In separate

experiments under the same conditions, the amounts of35

S-labelled gp120 retained in pellets of radioS-labelled extracellular virus after incubation with inactivating MAb were identical at the two temperatures as determined by radioimmunoprecipi-tation and sodium dodecyl sulfate-polyacrylamide gel electro-phoresis analysis.

Inactivation of primary and laboratory-adapted HIV-1 iso-lates.A panel of laboratory-adapted HIV-1 isolates (LAV, RF, and MN), a cloned primary isolate (JR-CSF), and five primary isolates from HIV-1-infected patients, grown in PBL, were tested with a panel of anti-HIV-1 human sera (Table 4). In general, laboratory-adapted strains were more susceptible to the inactivating effects of anti-HIV sera than were the primary isolates. In Table 4, sera and isolates with the same number are contemporary specimens from the same person. The inactivat-ing activity of sera for autologous isolates was even weaker than the inactivating activity of these same sera for unrelated primary isolates. In fact, sera that showed minimal activity with autologous isolates often had reasonably good activity for an-other patient’s primary isolate. In only one case, however, was there a reciprocal relationship (i.e., serum from patient 5151 was relatively inactive against its own isolate and active against isolate 73380, whereas serum 73380 was inactive against its own isolate and active against isolate 5151).

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The possibility that blocking antibodies were responsible for the poor inactivation of autologous isolates was approached in

TABLE 2. Inactivation of HIV-1LAIby monospecific antibodies

Antiserum or antibodya

Specificity

Assay resultb

H9 immunofluorescence 35S-labelled

gp120 RIP

Log titer reduction Cytoplasmic (1:100) Cell surface MFI

Mouse MAb 9303 gp41 1 51 0.00

Human MAb 50-69 gp41 1 48 0.45

Sheep polyclonal serum gp120 1 .3.56

Goat PB1 serum gp120, aac295–474 1 .3.06

Goat PB1sub7 serum gp120, aa 350–405 2.81

Goat PB1sub2 serum gp120, aa 295–404 3.06

Mouse MAb 9201 gp120, aa 475–486 1 0 2 0.00

Mouse MAb 110.1 gp120, C terminus 1 6 1 0.44

Mouse MAb 110.4 gp120, V3 region 1 113 1 3.04

Mouse MAb 110.5 gp120, V3 region 1 126 1 3.10

Mouse MAb 0.5b gp120, V3 region 1 123 1 3.06

Mouse MAb 9305 gp120, V3 region 1 123 1 2.89

Human MAb F105 gp120, CD4bd 1 92 1 2.23

Human MAb 15E gp120, CD4bd 1 60 1 1.50

aAll of the antisera and antibodies reacted with the test strain, HIV-1

LAI. For cytoplasmic staining and fluorescence microscopy of HIV-1-infected H9 cells, a 1:100 dilution was tested. For cell surface staining and cytofluorometry of H9 cells, serial dilutions of antibodies starting at 1:10 were tested. The maximum mean fluorescence intensity (MFI) minus that of the reagent control is shown. Radioimmunoprecipitations (RIP) of35S-labelled HIV-1

LAIdetergent lysates were performed with 10ml of antibody. For inactivation, antisera were tested at a 1:4 final dilution and MAbs were tested at 1:10. CD4bd, conformational determinant related to the CD4bd.

b

Sheep anti-gp120 serum (ARRRP no. 228) was obtained through the ARRRP, courtesy of M. Phelan (48). MAbs 9303, 9201, and 9305 were from Du Pont (13). MAb 50-69 was from the ARRRP (no. 531), courtesy of S. Zolla-Pazner (49). Goat antisera PB1 (no. 36), PB1Sub7 (no. 38), and PB1sub2 (no. 40) were from the ARRRP, courtesy of Repligen Corp. (28, 52). MAbs 110.1, 110.4, and 110.5 were from Genetics Systems Corp. (Seattle, Wash.), courtesy of E. Kinney-Thomas (23). MAb 0.5bwas from the ARRRP (no. 904), courtesy of S. Matsushita (28). MAb F105 was from the ARRRP (no. 857), courtesy of M. Posner (50). MAb 15E was from D. D. Ho (Aaron Diamond AIDS Research Center, New York, N.Y.) (20).

c

aa, amino acid.

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the following way. An isolate was mixed with autologous se-rum. A heterologous serum with good activity for the particu-lar isolate was then added (1:4 or 1:8 final concentration of each serum). The inactivation observed was compared to that of the heterologous serum plus nonimmune human serum (NHS) and autologous serum plus NHS. Three isolates (5151, 5157, and 73380) were tested in this way. In no case did the presence of autologous serum interfere with the inactivating activity of the heterologous serum. There also was no discern-ible blocking (or enhancing) effect of autologous serum when the heterologous serum was added in dilutions that were mar-ginally inactivating.

Heterologous inactivation of a reference HIV-1 strain (LAI) by serial sera from HIV-1-infected men. Three to five speci-mens collected at approximately yearly intervals from 15 HIV-1-infected homosexual men were tested for inactivation of a reference strain (LAI) of HIV-1 (Fig. 4). Eight of these men progressed to AIDS over the course of observation (progres-sors), and seven did not (nonprogressors). All of the men presented with generalized lymphadenopathy, and the time line for plotting of data was months since the onset of

lymph-adenopathy for both groups (Fig. 4, left and middle panels, respectively). For progressors, the same data were also plotted relative to the onset of an AIDS-defining clinical complication (panels on right). Nonprogressors had generally higher levels

of inactivating activity than did progressors (P50.0147,

Wil-coxin rank sum test), although activities in the initial specimens were not significantly different (Fig. 4a, b, and c). Similarly,

infectious virus titers were higher in progressors (P50.011)

and tended to rise progressively with time, but they were not significantly different when only the first specimens were com-pared (Fig. 4d, e, and f). Likewise, initial CD4 cell levels did not distinguish progressors from nonprogressors, but levels in progressors subsequently declined. Overall, absolute CD4 cell levels, percent CD4 cells, and rate of CD4 loss were all

signif-icantly different from those of nonprogressors (P50.0001 to

0.0292). Antibody titers to specific viral proteins determined by Western blot analysis were not significantly different between the groups, nor were absolute lymphocyte counts or absolute CD8 cell counts.

Inactivation of autologous isolates by serial sera from HIV-1-infected men.Virus isolates were obtained from serial sam-ples obtained approximately a year apart and tested in a check-erboard fashion with contemporary sera (Tables 5 and 6). Isolates tended to be resistant to inactivation by contemporary or earlier sera but were sensitive to later sera. Two patients, one of whom developed AIDS and one of whom did not, were tested. They showed similar patterns, in which virus isolates emerge that escape inactivation by earlier or contemporary sera.

Heteroduplex assay.Heteroduplex analysis (Fig. 5) was per-formed with material from the subjects presented in Tables 5 and 6 to assess (i) the relatedness of sequential isolates to each other, (ii) whether the isolates were representative of the virus species that existed in the subject’s PBL at the time of isola-tion, and (iii) changes in the relative abundance in PBL over time.

Isolates 1, 2, and 4 formed a prominent homoduplex band and a prominent, more slowly migrating heteroduplex band (or

closely migrating doublet bands) with their own 32P-labelled

DNAs, indicating two major viral species. Isolate 3 formed a single prominent homoduplex with its own DNA, indicating a more homogeneous virus isolate. These patterns of homolo-gous duplex formation were almost identical to the duplex

pattern obtained when32P-labelled isolate DNA was annealed

[image:5.612.58.299.73.420.2]

with DNA from contemporary PBL, the source of the respec-tive virus isolates. Thus, the virus we isolated at any time point

FIG. 3. Inactivation of HIV-1 infectivity in immunoprecipitates. HIV-1LAI (100ml) was incubated with an equal volume of anti-HIV-1 serum 5111 at the indicated final dilutions for 2 h at 08C. The preparations were centrifuged, and the pellets were resuspended in medium (100ml). (a) p24 content of precipitates. (b) Resuspended precipitates were further incubated at 378C (F) or 08C (■) for 2 h, at which time the preparations were diluted for determination of the infectious titer (50% infective dose). The titer of the starting material was 104.05, and the p24 content was 1,452 pg. The p24 content of a control precipitate formed with NHS at 1:4 was 145 pg.

TABLE 3. Supernatant gp120 dissociated from HIV-1LAIparticles

after a 2-h incubation at 0 or 378C

Incubation mixturea

(dilution or concn [mg/ml])

Temp (8C)

Amt (ng) of supernatant

gp120

Expt 1 Expt 2

HIV-11buffer 0 9 31

HIV-11buffer 37 28 67

HIV-11anti-gp120 V3 MAb (1:10)b 0 2 22

HIV-11anti-gp120 V3 MAb (1:10) 37 27 39

HIV-11sCD4 (50) 0 135 109

HIV-11sCD4 (50) 37 138 194

HIV-1 lysate1anti-gp120 V3 MAb (1:10)c 0 153 321

HIV-1 lysate1anti-gp120 V3 MAb (1:10) 37 174 354 a

Volume, 100ml.

b

Dupont MAb 9305.

c

The gp120 contents of viral lysates processed in the presence or absence of a MAb were similar.

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was highly related to the most abundant proviral sequences found in PBL at the same time. However, in cross-hybridiza-tion, these species were not the most abundant species found in PBL at earlier or later time points. The degree of

related-ness of isolate DNA with other isolate DNA or PBL DNA, assessed by migration of duplexes (the further the migration, the more related the species), is consistent with temporal evo-lution (the closer in time, the more related the species).

[image:6.612.65.554.84.191.2]

Het-FIG. 4. Serial studies of HIV-1-infected men who progressed or did not progress to AIDS. Serum inactivation of HIV-1LAI(a, b, and c), infectious virus titers (d, e, and f), and absolute CD4 T-cell levels (g, h, and i) were determined in HIV-1-infected men. (a, d, and g) Data for those who did not develop AIDS-defining conditions (nonprogressors) plotted relative to the onset of lymphadenopathy (the presenting symptom of all of the men). Data for men who subsequently developed AIDS-defining conditions (progressors) are presented relative to the onset of lymphadenopathy (b, e, and h), and the same data are plotted relative to the onset of AIDS (c, f, and i).

TABLE 4. Serum inactivation of HIV-1 isolatesa

Serum Log reduction in titer of HIV-1 isolate:

LAI RF MN JR-CSF 5151 5157 73380 81927 5084

5151 .4.44 .3.22 .3.86 .3.59 1.11 .3.39 2.22 .2.66

5157 0.78 .3.22 1.11 1.69 0.58 0.00 0.61 0.49

73380 4.19 .3.22 .3.86 .3.59 .2.88 .3.39 0.27 1.66

81927 2.11 0.63 0.61 0.00 0.05

5084 .3.00 0.39

5028 .4.00 .3.22 2.49 .3.59 1.43 1.72 1.94

5096 4.40 .3.22 1.12 1.73 0.29 1.38 0.48

5111 3.94 .3.22 2.83 .3.59 2.00 2.17 1.61 1.67 .2.94

aLAI, RF, and MN are laboratory-adapted isolates. JR-CSF was derived from a clone of a minimally passed primary isolate. The remainder are primary isolates.

All isolates were propagated in PBL. Sera and isolates with the same number are contemporary specimens from the same individual.

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eroduplex analysis was also performed on the patient reported in Table 6 with essentially the same results (data not shown).

DISCUSSION

In the inactivation assay, undiluted HIV-1 is preincubated at

378C with serum and an infectivity titration of the mixture is

performed (Fig. 1 and 2). The reduction in titer cannot be explained on the basis of reversible blockage of virus binding to target cells or neutralization that occurs in the culture. With dilution, the amount of serum actually present in the cultures is orders of magnitude lower than that required for these same sera in the conventional neutralization assay, and for most dilutions, the amount of virus present is greater. If the titer reduction were due to neutralization that occurs in culture, the

control cultures (preincubation of mixture at 08C or separate

incubation control) should register similar results. With serum in excess, binding to virions should be maximal within 2 h at

both 37 and 08C. We found incubation of serum for 2 h at 0 or

378C to be more than adequate for optimal and equivalent

binding of serum antibody to H9 cells and for maximal and equivalent immunoprecipitation of radiolabelled extracellular HIV-1. Furthermore, after preincubation of the virus-serum

mixtures at 37 or 08C, prolonged incubation of the serial

dilu-tions at 378C (or 08C) for 6 or 18 h before addition to the cells

(to establish similar equilibria) did not result in inactivation in

the sample preincubated at 08C nor did it reverse the

inacti-vation in the sample preincubated at 378C. The requirement

for preincubation at 378C could reflect the display of a cryptic

HIV-1 epitope at 378C that is not accessible at 08C, different

binding kinetics for a subset of antibodies, or a combination of the two (induced-fit reaction). These would be presumably minor, yet highly relevant, reactions that might be obscured by the polyspecific reaction of human sera in the H9 or immuno-precipitation assays. However, monospecific antibodies reac-tive with known epitopes inactivate without preferential or

more complete binding at 378C (Table 2). Furthermore, with

polyclonal sera, relevant binding does, in fact, occur at 08C.

Antibody-virus precipitates that are formed at 08C and have

had excess antibody removed by centrifugation are inactivated

by further incubation at 378C (Fig. 3). An alternative

explana-tion, the one we favor, is that the 378C incubation provides the

activation energy required for bound antibodies to shift the viral structure from a functionally infectious state to an inac-tive state. Precedence for an sCD4- or antibody-induced con-formational change in gp120 (9, 56, 68) and for other viruses, where the change results in functional impairment, has been demonstrated (4, 11, 18, 22, 69). We propose that antibody triggers, in the absence of target cells, an irreversible event that normally occurs during virus penetration. The penetration event itself (as opposed to the binding event) also requires a

378C incubation and does not occur at 4 or 08C (46).

There are several advantages to this system. Serum is as-sayed with undiluted virus, and this avoids the difficulty of consistently providing the small infectious dose (10 to 100 tissue culture-infective doses) required to obtain reproducible serum titers in the conventional neutralization assay. Most

neutralization assays do, however, incorporate a 378C

preincu-bation step (1, 2, 10, 12, 29, 35, 37, 42, 62, 66, 67), a feature we found critical for maximal inactivation. It could be argued that testing of relatively high concentrations of serum and virus, rather than limiting amounts, more closely approximates con-ditions in vivo. The inactivation assay is quite reproducible. The registered log reduction in titer of a given strain with a given serum varies by no more than 1 log from run to run, regardless of the titer of the virus stock (mean interrun

[image:7.612.318.552.68.355.2]

differ-FIG. 5. Heteroduplex assay of serial isolates. PCR-generated, 700-bp env-encoded,32P-labelled proviral DNAs from serial isolates 1 through 4 (a to d) from patient 5084 (Table 5) were annealed with serial specimens of PCR-generated PBL proviral DNAs (PBL DNAs 1 to 4) and with their respective HIV-1 isolate DNAs (isolate DNAs 1 to 4). Lane P,32P-labelled isolate DNA run alone. Asterisks mark the migration of single-stranded DNA, and arrows indicate the locations of homoduplex bands.

TABLE 5. Inactivation of serial HIV-1 isolates from subject 5084, a nonprogressor, by autologous sera

Serum sample

Log reduction in titer of HIV-1 isolate:

5084-4a

5084-21 5084-37 5084-50 LAI

5084-4 0.39 0.00 0.00 0.61 .3.00

5084-21 .2.94 1.39 0.00 0.61 .3.00

5084-37 .2.94 2.45 0.39 0.00 .3.00

5084-50 .2.94 2.64 .2.39 0.00 .3.00

5111 .2.94 1.78 .2.39 .2.00 .3.00

aThe number after the hyphen is the number of months since the onset of

[image:7.612.58.298.93.173.2]

lymphadenopathy.

TABLE 6. Inactivation of serial HIV-1 isolates from subject 5157, a progressor, by autologous sera

Serum sample

Log reduction in titer of HIV-1 isolate:

5157-4a 5157-13 5157-28 5157-37 LAI

5157-4 0.00 0.39 1.28 0.00 0.78

5157-13 .2.00 1.00 1.28 0.00 0.39

5157-28 .2.00 2.06 1.73 0.58 1.39

5157-37 .2.00 1.00 1.67 0.33 1.39

5111 2.17 1.00 .2.67 .2.83 .3.89

a

The number after the hyphen is the number of months since the onset of lymphadenopathy.

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ence in log titer reduction, 0.3260.32). We do not consider a less than 1.5-log (97% or 31-fold) reduction significant, whereas the end points for serum neutralization assays are generally reductions in virus production of 50 or 90%. Thus, differences in end point sensitivity and a more stringent infec-tivity threshold result in satisfactory reproducibility. However, we may well sacrifice a level of sensitivity for small or subtle effects of serum (or differences between sera) on virus infec-tivity. Another advantage to this assay is that the control and test cultures contain identical amounts of virus and serum, differing only in the manipulation of these components before their addition to culture. This effectively controls for any effect of residual serum on cells in culture or on the immunoassay used to monitor virus replication (27). Finally, the difference between control and inactivation culture titer results indicate an effect of serum on virus infectivity before exposure to target cells, and the assay lends itself to some experimental manipu-lations for examining the mechanism of antibody interference with infectivity.

Inactivation of HIV-1 requires preincubation at 378C with

relatively high concentrations of antibody-positive sera. If these conditions are met, inactivation appears to be irrevers-ible. We offer the following as evidence for the irreversibility of inactivation. First, and most simplistically, returning a

virus-antibody mixture preincubated at 378C to 08C does not restore

infectivity. Second, despite the different manipulations of an-tibody and virus before addition to culture, the inactivation and control cultures contain identical amounts of virus and antibody and would be expected to ultimately arrive at the same virus-antibody-cell equilibrium. If the antibody effect on infectivity is reversible, these cultures should register similar titers, and they do not (Fig. 1). Finally, the infectivity of virus

washed free of unbound antibody and inactivated at 378C is not

restored by allowing virus dilutions to sit at 378C (or 08C) for

18 h before addition to cell culture. Excess dilution after re-moval of unbound antibody is the principal means by which off rates of antigen-antibody reactions are measured (63).

An assay similar in format was used by Spear et al. (61) to measure complement-mediated lysis-agglutination of HIV-1, although the activity we measured is not dependent on

com-plement. No evidence of a Ca21- or Mg21-dependent or

heat-labile cofactor in human serum was obtained, and IgG purified from serum mediated the effect on its own (Table 1). Bivalent

F(ab9)2 fragments of anti-HIV-1 IgG were active, although

perhaps somewhat less so than intact IgG. Monovalent Fab fragments, tested in equibinding concentrations, were not ac-tive (Table 1). Several studies with anti-HIV monoclonal Fab fragments indicate that some fragments retain neutralizing ac-tivity whereas others do not (3, 5, 53). This suggests that dis-tinct bivalence-dependent and monovalent mechanisms of neutralization exist.

The small panel of monospecific antibodies was used to address two questions. First, does reactivity with a single epitope mediate inactivation? Second, is there a concordance or discordance between epitopes that are neutralizing deter-minants and those that are inactivating deterdeter-minants? Anti-body reactivity with a single epitope is sufficient to mediate inactivation (Table 2). Two epitopes, the V3 region of gp120 and a conformation-dependent epitope(s) related to the CD4-binding domain (CD4bd) of gp120, both well-established neu-tralizing determinants in conventional neuneu-tralizing assays (20, 50, 53, 54), were also targets for inactivation in our assay (Table 2). All monospecific reagents reacted with the test stain of HIV-1 in one or more immunoassays. The one immunoassay that was predictive of inactivation was reactivity with cell sur-face gp120. It seems reasonable that access to epitopes in their

native configuration on intact virus, for which cell surface dis-play is a reasonable model, would be a prerequisite for anti-body interference with infectivity. Moore et al. reported an absolute concordance between neutralization and reactivity with the cell surface oligomeric envelope (37, 41, 57). They further proposed that additional, unidentified determinants, unique to the oligomeric structure, may be potent and poten-tially important targets of neutralization (36, 38, 57).

The mechanism of inactivation does not appear to be agglu-tination of virus or induced shedding of gp120 from virions. When virus was purposely agglutinated by

immunoprecipita-tion at 08C, infectious titers were recovered from the

precipi-tate. The titer was abolished by further incubation at 378C.

Furthermore, the optimal serum dilutions for immunoprecipi-tation (agglutination) and inactivation were different (Fig. 3). Since the sum of infectious virus in the immunoprecipitate plus its supernatant was less than that of the starting virus prepa-ration, it is apparently possible to reduce the titer by aggluti-nation. Physical packing of virions by centrifugation may be required to optimally demonstrate it. In this regard, an

occa-sional serum registers a lower titer in the 08C control than

either the separate dilution control or the virus titration in the absence of antibody (although it is never as low as that of the

mixture incubated at 378C and can be further reduced by a

378C incubation). When there is a titer reduction at 08C,

ag-glutination may be responsible.

When radiolabelled extracellular virus was incubated with

anti-HIV-1 serum at 37 or 08C for 2 h in physiologic buffer and

subsequently processed at 08C for radioimmunoprecipitation,

we detected no difference in either the amount or profile of the

viral proteins precipitated. We found no evidence that the 378C

incubation induced shedding of gp120 in an assay measuring residual envelope expression of H9 cells after exposure to antibody. Nor did an inactivating MAb induce any substantial release of envelope into the supernatant after pelleting of virus particles (Table 3). The latter experiments were, of necessity, performed with MAbs rather than with polyclonal sera. Be-cause of the functional parallelism between the MAb and poly-clonal sera in the inactivation assay, we presume that the mechanistic study of a MAb would apply to most, if not all, reactivities in polyclonal sera as well.

Heterologous inactivation (serum and virus isolate from dif-ferent sources) was generally better against laboratory-adapted isolates than against primary patient isolates (Table 4). A sim-ilar difference has been noted in conventional neutralization assays (12, 29, 37, 67). The basis for this difference is not clear but may relate to intrinsic biologic properties of the virus. Primary isolates often differ from laboratory isolates in other properties as well: sensitivity to sCD4, cell tropism, and syncy-tium formation (6, 7, 19, 45). Heterologous inactivation for a reference strain (LAI) was tested in serial specimens from homosexual men, of whom some progressed to AIDS and others did not (Fig. 4). Progression was significantly associated with lower levels of inactivation. Conventional neutralization (in our study) was not, although there was a correlation

be-tween the two assays (r 50.69, P, 0.001). Progression was

also significantly associated with rising virus titers and declin-ing CD4 cell levels. However, when only results from the first sample were analyzed, none of the assays were predictive of progression. We have not looked for other clinical associations with heterologous inactivation because it is apparent that het-erologous inactivation is a poor predictor of inactivation for autologous isolates. The latter is likely more relevant.

Relatively poor neutralization of autologous isolates has been reported by several groups (1, 2, 10, 35, 42, 62, 66). It is perplexing that autologous inactivation was often much poorer

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than heterologous inactivation of the same primary isolate by unrelated sera (Tables 4 to 6). We found no evidence of anti-bodies in autologous sera that block inactivating activity. It appears that selection of virus isolates is custom tailored, as it were, for resistance to autologous immune attack. Heterolo-gous inactivation is often reasonably potent, and later sera often have better activity against earlier autologous isolates than do heterologous sera. Both of these facts reflect antigenic plasticity on the part of the virus and a remarkable and effective, albeit delayed, accommodation on the part of the immune system. Several investigators have reported escape from neutraliza-tion by serially obtained autologous isolates (1, 2, 10, 35, 42, 62, 66). The serial inactivation studies reported here followed the same pattern (Tables 5 and 6). To obtain an autologous isolate for testing, one must necessarily culture virus, and the virus that emerges from coculture (where additional and irrelevant selection pressures may occur) may not be entirely represen-tative of the viral species replicating in the host at the time (34). On the other hand, it has been concluded, on the basis of sequence analysis, that virus isolates obtained from short-term culture are representative of the infectious species that exist at the time of isolation in both PBL and plasma (55). Heterodu-plex mobility assays indicate that the virus species we isolated were highly related to the most abundant PBL proviral species present at the time, and these species were minor species at earlier and later time points (Fig. 5). The question then be-comes: how representative is PBL proviral DNA of actively replicating virus or of virus found in various lymphoid com-partments? If one reasonably assumes that plasma virus rep-resents currently replicating virus (21, 64), the lag time be-tween the appearance and predominance of a new virus species in plasma and its appearance and predominance in PBL pro-viral DNA can be as short as 14 days (64), although 50 to 100 days is probably a better estimate (24, 58, 59). Since PBL recirculate between lymphoid compartments, it seems likely that, to some extent, their proviral sequences reflect the virus species replicating in those compartments. Thus, PBL proviral sequences can be considered a catalog reflecting, for the most part, the host’s experience with replicating virus over the past 2 to 3 months. If so, there appears to be a dynamic and causal relationship between the abundance of viral species as mea-sured by heteroduplex assay and the recognition (elimination) or nonrecognition of viral species as measured by serum inac-tivating activity.

It remains to be determined what the importance of a neu-tralization assay conducted in the inactivation format de-scribed here will be. Most neutralization assays incorporate the

important conditions emphasized here (378C preincubation

with relatively high concentrations of sera) but differ in the mechanics and details for measuring reduction in virus repli-cation. It remains to be shown whether any assay accurately reflects the correlates of protective or disease-controlling im-munity by antibody. In any event, the fact that envelope en-gagement by antibody, as well as by an analog of its natural ligand (sCD4), results in temperature-dependent inactivation of infectivity implies that a critical structural conformation or conformational change in the envelope is required for virus entry. Gaining insight into the mechanism of antibody inter-ference with viral infectivity, the structural requirements of virus for entry and successful infection, and the access or vul-nerability of the requisite structure or structural change to antibody may contribute enormously to the quest for an effec-tive immune intervention in HIV disease.

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Figure

FIG. 1. Inactivation of HIV-1 by anti-HIV-1 serum. An undiluted HIV-1stock was mixed with anti-HIV-1 serum 5111 (1:4 final serum dilution) andLAI
TABLE 1. Inactivation of HIV-1 by serum components or fractions
TABLE 2. Inactivation of HIV-1LAI by monospecific antibodies
TABLE 3. Supernatant gp120 dissociated from HIV-1LAI particlesafter a 2-h incubation at 0 or 37�C
+3

References

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