Vol. 66, No. 9
JOURNAL OF VIROLOGY, Sept. 1992, p. 5210-5215
0022-538X/92/095210-06$02.00/0 Copyright © 1992, American Society for Microbiology
Examination of Sera from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals for Antibodies Reactive with Peptides Corresponding to the Principal Neutralizing Determinant of HIV-1 gpl20 and for In Vitro Neutralizing Activity RONALD Q. WARREN,1'2 STEPHANIE A. ANDERSON,1'2 WATOKY M. M. M. NKYA,3 JOHN F. SHAO,4 CRAIG W. HENDRIX,2'5 GREGORY P. MELCHER,2'5 ROBERT R. REDFIELD,6 AND RONALD C. KENNEDY' 2* Department of Virology and Immunology1 and Center for AIDS Research,2 Southwest Foundation for Biomedical Research, San Antonio, Te-xas 78284-0147; Kilimanjaro Christian Medical Center, Moshi, 3 and Muhimbili Medical Center, Dar es Salaam, 4 Tanzania; Department of Medicine, Wilford Hall Medical Center, Lackland Air Force Base, Texas 782365; and Department of Retroviral Research, Walter Reed Army Institute of Research, Rockville, Maryland 208506 Received 10 January 1992/Accepted 1 June 1992
Sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals from the United States and Tanzania were examined for antibody reactivity to four synthetic peptides which corresponded to the principal neutralizing determinant from the V3 region of HlV-1 gpl20. We observed that the majority of sera from both countries contained antibodies reactive with a V3 peptide whose sequence is based on that of the HIV-1 MN isolate. We were unable to establish a relationship between the presence of V3-reactive antibodies, as measured by enzyme-linked immunosorbent assay and neutralization of homologous HIV-1 isolates, in sera from either the United States or Tanzania. We observed that some sera which contained high antibody titers to the V3 peptides failed to neutralize HIV-1, while others with no antibody reactivity to the panel of V3 peptides exhibited in vitro neutralizing activity. These results suggest that neutralizing epitopes exist outside the V3 loop and that the presence of V3-reactive antibodies in sera does not imply in vitro neutralization of the homologous HIV-1 isolate. In addition, it appears that the V3 loop may consist of both neutralizing and nonneutralizing epitopes. The identification of neutralizing as well as nonneutralizing epitopes will be important for the design of potential HIV-1 vaccines. The principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) is thought to consist of a disulfide-linked loop structure spanning the third variable region (V3) of gpl20 (18). This immunodominant epitope corresponds to amino acid residues 303 to 338 of gpl20 (IIIB) (40) and has been reported to induce type-specific neutralizing antibodies in vitro (11, 12, 22, 37, 38). On the basis of the induction of neutralizing antibodies in experimental animals, this viral epitope is under investigation as a candidate for potential HIV-1 vaccines and as a target for immunoprophylactic strategies (8, 9, 33). Thus, characterization of the immune response to this epitope following natural infection
from infected individuals by enzyme-linked immunosorbent assay (ELISA) and their ability to neutralize homologous HIV-1 isolates in vitro. This lack of correlation between antibodies to V3 epitopes defined by synthetic peptides and neutralizing activity suggests that conformational epitopes associated with V3 or non-V3 epitopes may play an important role in the neutralization of HIV-1 among infected individuals.
MATERIALS AND METHODS Human sera. Serum specimens from HIV-1-infected individuals from the United States (n = 37) and Tanzania (n = 74) were examined in this study. All specimens from HIV1-infected individuals were confirmed to contain anti-HIV-1 gpl60 antibodies by Western immunoblot analysis (Bio-Rad Laboratories, Richmond, Calif.). Serum specimens from the United States were obtained from U.S. military personnel. Because of the limitations in the quantity of sera available from HIV-1-infected individuals from Tanzania, 15 serum samples were selected to be characterized in the neutralization assays. Ten serum samples from healthy, uninfected individuals were used as controls. All serum samples were heat inactivated at 56°C for 30 min prior to use. Synthetic V3 region peptides. Peptides RP135, RP142, and RP145, which correspond to the gpl20 V3 region of HIV-1
with HIV-1 in humans from geographically diverse populations is important for the design and effectiveness of these potential strategies for controlling HIV-1 infection. In this study, we examined sera from HIV-1-infected individuals from both the United States and Tanzania for antibodies reactive with four synthetic peptides whose sequences are based on the V3 region. Specifically, we compared sera for antibody reactivity to the V3 peptides together with their ability to neutralize HIV-1 isolates in vitro. The results described herein indicate that there is little correlation between the detection of V3-reactive antibodies in sera *
Corresponding author. 5210
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TABLE 1. Amino acid sequences of principal neutralizing determinant-derived V3 synthetic peptides used in this study
Peptide
(amino acid residues) 304-321 RP135 (308-331) RP142 (306-330) RP145 (303-325)
HIV-1
Amino acid
isolate
IIIB IIIB
sequence0
(CGY)TRPNNNTRKSIRIQRGPG NNTRKSIRIQRGPGRAFVTIGKIG(C)
MN
YNnKBRIHIGPGRAFYTTKNIIG(C)
HAN/SC
NNTRKGIHIGPGRA/FYATGDIIG(C)b
a Amino acids in parentheses were added to facilitate coupling to carrier proteins for related immunization studies. b The slash mark indicates division between HAN and SC amino acid sequences.
isolates IIIB (LAI) (42) and MN and the composite HAN/ SC, respectively, were kindly supplied by Scott Putney (Repligen, Cambridge, Mass.). The composite V3 peptide, RP145, was examined to assess whether hybrid sequences could be recognized by antibodies from naturally infected individuals (17). Peptide 304-321, corresponding to the IIIB isolate, was synthesized by methods previously described (43). The amino acid sequences of the four V3 region-derived peptides are depicted in Table 1. Determination of antibody binding to synthetic peptides as measured by ELISA. The methods for detecting antibody reactivity to HIV-1 gpl60 synthetic peptides by ELISA have been described in detail previously (5, 21, 43). All antisera (1/50 dilution) were screened for V3-reactive antibodies in at least three separate determinations. Ten HIV-seronegative normal control sera were similarly tested for antibodies reactive to the synthetic peptides. Experimental optical density (OD) values greater than three times the mean OD value obtained for the 10 control sera were considered positive in reactivity for a given peptide. This positive reactivity cutoff level for these sera exceeded the 95% confidence level. In those instances when normal control sera resulted in a negligible OD value and a cutoff value for positive reactivity could not be calculated, an OD value of 0.1 was used as the positive cutoff. Endpoint antibody titers of sera were determined to be the greatest dilution of sera which resulted in an OD value greater than the positive cutoff. Neutralization assays. Two neutralization assays were performed in this study. In the first assay, sera to be examined were preincubated for 1 h with 100 50% tissue culture infective doses of HIV-1 isolate IIIB. This mixture (50 ,ul) was then added to microtiter wells containing 25,000 SupTl cells (100 ,ul, final volume). Cells were incubated at 37°C for
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8 days, being fed on days 3 and 6 with RPMI containing 15% fetal bovine serum. On day 8, 10 ,ul of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; thiazolyl blue; 0.012 M; Sigma, St. Louis, Mo.) was added to the individual wells for 4 h at 37°C. The cultures were then inactivated by the addition of 175 ,ul of 0.04 N HCI in isopropanol, and the OD at 570 nm was determined. Endpoint neutralization titers were calculated as the greatest dilution of sera which resulted in 50% target cell viability in comparison with control wells containing normal sera (34). The MTT neutralization assay was performed only with the IIIB isolate, since the MN isolate was not cytotoxic toward the SupTl target cells. In the second assay, a p24 antigen capture ELISA was used to determine neutralizing activity of sera against both the IIIB and MN isolates (100 50% tissue culture infective doses) (Retrovirology Coulter Corp., Hialeah, Fla.). The cells and virus were cultured as described above, with the HIV-1 p24 levels in culture supematant being determined on day 8 according to the manufacturer's recommended procedure. Sera were considered to contain neutralizing activity if greater than 50% reductions in p24 levels were detected in comparison with wells containing normal sera at a 1/50 dilution. Neutralization endpoint titers were determined as described above. Because of the variation in target cell susceptibility to infection by various HIV-1 isolates, we also examined CEM-SS cells as target cells in the neutralization assays. The p24 ELISA was used to assess neutralization of both the IIIB and MN isolates with the CEM-SS target cell line. Briefly, 50,000 CEM-SS target cells were adhered onto a poly-L-lysine-coated microtiter plate. Following a 1-h incubation at 25°C, 50 ,ul of the virus-serum mixture was added to the target cells, and the mixture was allowed to incubate for 8 days at 37°C. Supernatants were harvested and screened for p24 reactivity as described above. Statistical analysis. Sera were examined for the significance of peptide-reactive antibodies and neutralizing activity by the chi-square test (35). In addition, correlation coefficients (r values) were calculated to compare antibody titers, as assessed by ELISA, and in vitro neutralization titers.
RESULTS
Serum specimens from HIV-1-infected individuals from the United States and Tanzania were examined for antibody reactivity to four V3-based synthetic peptides. These specimens were also examined for neutralizing activity against HIV-1 isolates IIIB and MN (Table 2). Peptide 304-321, which corresponds to the amino half of the V3 loop of the
TABLE 2. Summary of serum reactivity from HIV-1-infected individuals to peptides corresponding to the V3 region of gpl20 and neutralizing activity No. (%) of sera with neutralizing No. (%) ofagainst of sera reactive to V3 peptide: HIV-1 isolate: activity serumN. (%
Source of serum
Source of
samples
IIIB 304-321
RP135
RP142
MN
RP145 p24
MIT
p24
5 (14) 10 (27) 31 (84)a 19 (51) 4 (11) 21 (57) United States (n = 37) 6 (16) 14 (19) 15 (20) 48 (65)39 (53) Tanzania (n = 74) 7 (47) 3b (27) 11C (73) a < Represents a statistically greater percentage of serum reactivity to RP142 than to RP135 (P 0.001). b Eleven representative serum samples from Tanzania were examined for neutralizing activity by p24 activity against the IIIB isolate. c Fifteen representative serum samples from Tanzania were examined for neutralizing activity by MTT uptake against the IIIB isolate and by p24 activity
against the MN isolate.
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WARREN ET AL.
IIIB isolate, was reactive with 5 of 37 serum specimens (14%) from the United States and 14 of 74 specimens (19%) from Tanzania by ELISA. Peptide RP135, which corresponds to the central portion of the V3 loop of the IIIB isolate, was reactive with a higher percentage of U.S. specimens (10 of 37 [27%]) than was peptide 304-321. Similar percentages of HIV-1-infected specimens from Tanzania were reactive with peptides RP135 (20%) and 304-321 (19%). Peptides RP142 and RP145, which correspond to the MN and HAN/SC isolates, respectively, were reactive with the majority of serum specimens examined from both countries. We observed that 31 of 37 serum specimens (84%) from the United States and 48 of 74 specimens (65%) from Tanzania were reactive with RP142. Nearly equal percentages of specimens from both countries were reactive with RP145 (51% from the United States and 53% from Tanzania). A statistically greater percentage of serum specimens from both the United States and Tanzania contained antibodies reactive with RP142 than with RP135 (P > 0.001), suggesting the widespread prevalence of HIV-1 MN-like isolates in both countries. Next, we examined these same serum specimens for their ability to neutralize HIV-1 isolates IIIB and MN in vitro and compared this activity with peptide ELISA reactivity. Two separate assays were used to determine neutralizing activity of sera against the IIIB isolate. These two assays measure different aspects of viral neutralization, since the p24 capture ELISA indicates the level of viral replication, while the MTT uptake assay measures the amount of target cell cytotoxicity by HIV-1. As seen in Table 2, a high percentage of serum specimens from both countries neutralized the IIIB isolate when assessed by the cellular cytotoxicity assay. We observed that 21 of 37 serum specimens (57%) from the United States and 11 of 15 serum specimens (73%) from Tanzania contained neutralizing activity. In contrast, fewer specimens exhibited neutralizing activity against IIIB when levels of p24 were measured in the culture supernatants. It therefore appears that relatively low levels of virus may lead to elevated p24 levels and no in vitro neutralizing activity as assessed by this method. However, under these conditions, target cell viability can remain high in vitro, indicating that a given serum sample may exhibit neutralizing activity, as judged from inhibition of HIV-i-induced cytotoxicity. These results indicate that care must be taken in interpreting serum neutralizing activity according to which assay is used. Individual serum samples from the United States were further characterized for antibody reactivity to the panel of V3 peptides and examined for levels of neutralizing activity against IIIB and MN (Table 3). These samples were examined to determine the importance of V3-reactive antibodies in sera and their effect on neutralizing activity against homologous HIV-1 isolates in vitro. Again, serum neutralizing activity against the IIIB isolate was measured by both the p24 antigen capture ELISA and MTT uptake. We were unable to establish a correlation between the presence of V3 peptide-reactive antibodies in sera from these individuals and viral neutralizing activity by using either neutralization assay. For example, we found no significant correlation between antibody reactivity to the IIIB-based V3 peptide RP135 and neutralization of IIIB on the basis of reduction of HIV-1 p24 (P = 0.617) or MTT uptake (P 0.104). In addition, of the 26 serum samples which lacked antibodies to either IIIB-based peptide (304-321 or RP135), 17 exhibited neutralizing activity (MTT) against the IIIB isolate in vitro. We also observed that of the 31 serum samples containing =
TABLE 3. Comparison of antipeptide reactivity and in vitro HIV-1 neutralizing activity of sera from infected individuals from the United States Neutralization titer
Antibody reactivity Serumody Serum
reactlvlty
against HIV-1 IIIB
sample 304-321
RP135
RP142
RP145
p24
+ 1 Negb + + 2 Neg + + Neg 3 + Neg 4 + 5 Neg + + + 6 Neg + + 100 + 7 8 Neg + + 50 9 + 200 10 + 11 Neg + + + 12 Neg + + + + 13 Neg + 14 Neg Neg 15 + 16 Neg + + + 17 Neg + + + + 18 Neg + + + + 19 Neg + + 50 20 21 Neg + + + 22 Neg + + + 23 Neg + + 24 Neg 25 Neg + 26 Neg + 27 Neg + + 28 Neg + 29 Neg + 30 Neg + 31 Neg + 32 Neg + + Neg 33 + + + 34 Neg + + Neg 35 + 36 Neg + + 37 Neg a Reciprocal dilution of serum that either reduced p24
isolatesa: MN
MiTT
(p24) ('4
Neg
Neg
50 Neg 50 100
Neg 100 100
Neg 800 100 50 200
50 Neg Neg 400 Neg 200 Neg 100 400 50 800 Neg Neg 200 Neg Neg Neg Neg 50 Neg 50 50 Neg Neg 50 50 200
100
Neg Neg Neg Neg
Neg Neg 200
Neg 200
Neg Neg Neg Neg Neg > 1,600 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg
levels by 50% or resulted in 50% target cell viability. b Neg, no neutralizing activity was detected at a 1/50 dilution of serum.
antibodies to peptide RP142, only 6 neutralized the homologous MN isolate in vitro. A similar pattern of neutralization was observed with 15 selected serum samples from HIV-1-infected individuals from Tanzania, as was previously seen with U.S. samples (Table 4). Again we found that a higher percentage of samples neutralized the IIIB isolate (11 of 15 [73%]), as measured by MTT uptake, than contained antibodies reactive with either of the IIIB-based peptides 304-321 (0 of 15) and RP135 (5 of 15 [33%]). In addition, we observed that six of these samples neutralized the IIIB isolate by the MTT assay yet lacked antibody reactivity to either of the IIIBbased V3 peptides. More importantly, we found that a high percentage of Tanzanian serum samples contained antibodies reactive with RP142 (13 of 15 [87%]), while only 7 (47%) neutralized the MN isolate in vitro (P = 0.509). As with the U.S. samples, we observed no significant correlation between the presence of V3-reactive antibodies and neutraliz-
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ANTIBODY REACTIVITY TO V3 PEPTIDES
TABLE 4. Comparison of antipeptide reactivity and in vitro neutralizing activity of sera from HIV-1-infected individuals from Tanzania
Neutralization titer
Antibody reactivity to V3 peptide: Serum sample
IIIB 304-321
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
RP135
-
+ -
-
-
-
+ + + +
-
-
-
-
RP142
+ + + + + + + + + + + + +
RP145
+ + + + + + + + + + + + +
MN
p24
MIT
p24
NDa ND 50 Negb 50 Neg Neg Neg ND Neg Neg Neg Neg 200 ND
800 800 100 800 200 400 1,600 800 400 Neg 400
100 200 50 Neg 50 Neg Neg Neg > 1,600 Neg
Neg Neg Neg 400
Neg Neg Neg 200 > 1,600
ND, because of the limited availability of some serum samples from Tanzania, the neutralizing activity as assessed by this assay was not determined. b Neg, no neutralizing activity was detected at a 1/50 dilution of serum. a
ing activity in sera from HIV-1-infected individuals from Tanzania. Since HIV-1 isolates can vary significantly in their target cell tropism, we examined 10 serum samples from the United States for neutralizing activity, using two target cell lines, SupTl and CEM-SS. Similar neutralization titers were observed when sera were tested against either the SupTl or CEM-SS target cells (data not shown). Thus, low levels of serum neutralizing activity toward the MN isolate, compared with high levels of reactivity to RP142, were not influenced by the target cells used in the neutralization assays. We then examined 14 selected serum samples from both countries to determine whether antibody titers to the V3 region peptides would correlate with neutralizing titers against homologous HIV-1 isolates. We were interested in determining whether high antibody titers to RP142 would lead to similarly high levels of neutralizing antibodies against the MN isolate. As seen in Fig. 1, no significant correlation was
56004800 t 4000
t
aN
3200w
Q-
2400
t
1600 '
t
8001 0
50
100
150
200
250
Neutrolization Titer (MN)
FIG. 1. Correlation between antibody endpoint titers to RP142 in sera from HIV-1-infected individuals and neutralizing titers against HIV-1 MN in vitro.
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observed between antibody titers to RP142 and neutralization titers of MN in vitro (r = 0.086). Similar results were observed with sera containing antibodies to RP135 and neutralization titers against the IIIB isolate (r = 0.138) (data not shown). DISCUSSION The V3 loop region of gp120 represents an immunodominant site on the envelope of HIV-1 which elicits type-specific neutralizing antibodies in immunized animals (12, 16, 19, 22, 29, 32, 33, 36, 38, 39). Deletion of the V3 loop region from gpl60 has been reported to result in a truncated glycoprotein that is unable to stimulate neutralizing antibodies in immunized animals (18). Previous studies have suggested that the majority of HIV-1 neutralizing antibodies are directed against the V3 loop. For example, preincubation of peptides RP135 or RP142 with HIV-1 antisera reportedly removes all neutralizing antibodies (18, 37). Amino acid substitutions within the V3 region of HIV-1 have been reported to lead to the emergence of neutralization-resistant variants (24-27). In addition, several studies have shown that the neutralizing activity of sera from HIV-1-infected individuals can be blocked by preincubation with V3 peptides (8, 44). However, additional HIV-1 neutralizing epitopes lying outside the V3 region of gpl20 have also been identified (1, 10, 14, 16, 20, 28, 30, 41). It is therefore important to understand the relationship between the detection of anti-V3 antibodies in sera from naturally infected individuals and the neutralization of HIV-1 in vitro. In this study, we were unable to establish a correlation between the presence of V3-reactive antibodies in sera and the neutralization of homologous HIV-1 isolates in vitro. For example, of 31 serum samples from the United States which contained antibodies reactive with RP142, only 6 neutralized the homologous MN isolate in vitro. We also found little correlation between the presence of antibodies to peptide 304-321 or RP135 and the ability of sera to neutralize IIIB. Carrow and colleagues reported a similar lack of correlation between the presence of anti-V3 antibodies in sera and neutralization of the IIIB isolate (4). However, these investigators did observe a significant correlation between the presence of anti-V3 antibodies and neutralization of the MN isolate. Our results also differ from those of Zwart and colleagues, who described a correlation between a single human serum sample containing V3-reactive antibodies and neutralizing activity in vitro (44). By using two separate neutralization assays, we observed quite different levels of serum neutralizing activity against the IIIB isolate. These differences can be attributed to the fact that the p24 antigen capture ELISA reflects the level of virus in the culture supernatant, while the MTT dye uptake assay measures the degree of cellular cytotoxicity in vitro. Our data indicate that while certain sera may protect target cells against HIV-1-mediated cytotoxicity, low levels of viable HIV-1 may remain in these cultures, with no neutralizing activity being observed when assessed by the HIV-1 p24 assay. Alternatively, the assay based on inhibition of cellular cytotoxicity may be more sensitive than the assay for reduction of HIV-1 p24. We observed that a high percentage of serum samples from both the United States and Tanzania contained antibodies reactive with the RP142 peptide. These results agree with those of earlier studies suggesting that the MN isolate, or antigenically related isolates, is commonly found worldwide (4, 6, 22, 44). In contrast, two peptides whose sequences correspond to that of the IIIB isolate (304-321 and
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WARREN ET AL.
RP135) were reactive with fewer than one-third of serum samples from either the United States or Tanzania. This low level of serum reactivity agrees with previous reports suggesting that the IIIB isolate currently represents a minor population of HIV-1 worldwide (6). Peptide RP135 appears to express additional epitopes in comparison with peptide 304-321, since a higher percentage of HIV-1-infected serum samples from the United States reacted with RP135. Peptide RP145, which corresponds to a composite of two HIV-1 isolates (HAN and SC), was reactive with approximately one-half of the Tanzanian and U.S. serum samples tested. We also examined whether serum endpoint titers to the V3 peptides would influence neutralization titers against homologous HIV-1 isolates. We were unable to establish a significant correlation between antibody titers to V3 peptides by ELISA and neutralizing activity. The presence of high antibody titers to RP142 (>1,600) had little effect on whether individual sera would neutralize the MN isolate in vitro. A similar lack of correlation between antibody titers to HIV-1 and neutralizing activity in vitro has been previously reported (31). Together, these results suggest that neutralizing epitopes lying outside the V3 region of gpl20 may play an important role in HIV-1 neutralization in vitro. The importance of target cells on serum neutralizing activity was also examined. Neutralization titers of sera appeared similar when two target cell lines were used (data not shown). Few sera neutralized the MN isolate when either the SupTl or CEM-SS cells were used as targets. These results further indicate that while most sera contained RP142-reactive antibodies by ELISA, few demonstrated neutralizing activity against the MN isolate in vitro. The observation of sera which contain V3-reactive antibodies yet lack neutralizing activity suggests that nonneutralizing epitopes may reside within the V3 loop. Antibodies reactive with these epitopes may demonstrate V3 reactivity by ELISA while exhibiting no neutralizing activity in vitro. A study by D'Souza and colleagues, using monoclonal antibodies specific for V3 epitopes, similarly found that antibody reactivity to V3 peptides did not equate with neutralizing activity (7). The amino acid sequence GPGR has been identified as an important target for in vitro neutralizing antibodies (2, 3, 23). Our study indicates that the majority of HIV-1-infected individuals do not produce antibodies reactive with this particular V3 epitope. The data presented here agree with those of previous studies suggesting that conformational V3 epitopes, along with epitopes lying outside the V3 region, play important roles in the neutralization of HIV-1 (13, 15). Our study indicates that the presence of antibodies reactive with V3-based synthetic peptides does not imply that sera will neutralize homologous HIV-1 isolates in vitro. In addition, we identified sera which lacked detectable anti-V3 antibodies by ELISA yet neutralized homologous HIV-1 isolates at high titers. Neutralizing sera which lack V3-reactive antibodies have been described recently by other investigators (2). It therefore appears that neutralizing epitopes which lie within the V3 region, along with those which lie outside it, will play equally important roles in potential vaccine development. In addition, our data suggest that the fine specificity of the humoral immune response to the V3 region may differ between experimentally immunized animals and naturally infected humans. Since other investigators have demonstrated that animals immunized with either RP135 or RP142 produce type-specific neutralizing antibodies, it appears that the V3 loop may contain neutralizing as well as nonneutralizing epitopes (18).
J. VIROL.
ACKNOWLEDGMENTS This work was supported by grants A126462 and A128696, a contract from the U.S. Army Research and Development Command, and the U.S. Agency for International Development. REFERENCES 1. Berkower, I., D. Murphy, C. C. Smith, and G. E. Smith. 1991. A predominant group-specific neutralizing epitope of human immunodeficiency virus type 1 maps to residues 342 to 511 of the envelope glycoprotein gp120. J. Virol. 65:5983-5990. 2. Broliden, P.-A., K. Ljunggren, J. Hinkula, E. Norrby, L. Akerblom, and B. Wahren. 1990. A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization. J. Virol. 64:936-940. 3. Broliden, P. A., B. Makitalo, L. Akerblom, J. Rosen, K. Broliden, G. Utter, M. Jondal, E. Norrby, and B. Wahren. 1991. Identification of amino acids in the V3 region of gpl20 critical for virus neutralization by human HIV-1-specific antibodies. Immunology 73:371-376. 4. Carrow, E. W., L. K. Vujcic, W. L. Glass, K. B. Seamon, S. C. Rastogi, R. M. Hendry, R. Boulos, N. Nzila, and G. V. Quinnan, Jr. 1991. High prevalence of antibodies to the gpl20 V3 region principal neutralizing determinant of HIV-1 MN in sera from Africa and the Americas. AIDS Res. Hum. Retroviruses 7:831838. 5. Chanh, T. C., G. R. Dreesman, P. Kanda, G. P. Linette, J. T. Sparrow, D. D. Ho, and R. C. Kennedy. 1986. Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO J. 5:3065-3071. 6. Devash, Y., T. J. Matthews, J. E. Drummond, K. Javaherian, D. J. Waters, L. 0. Arthur, W. A. Blattner, and J. R. Rusche. 1990. C-terminal fragments of gp120 and synthetic peptides from five HTLV-III strains: prevalence of antibodies to the HTLVIII-MN isolate in infected individuals. AIDS Res. Hum. Retroviruses 6:307-316. 7. D'Souza, M. P., P. Durda, C. V. Hanson, G. Milman, and collaborating investigators. 1991. Evaluation of monoclonal antibodies to HIV-1 by neutralization and serological assays: an international collaboration. AIDS 5:1061-1070. 8. Emini, E. A., P. L. Nara, W. A. Schleif, J. A. Lewis, J. P. Davide, D. R. Lee, J. Kessler, S. Conley, S. Matsushita, S. D. Putney, R. J. Gerety, and J. W. Eichberg. 1990. Antibodymediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees. J. Virol. 64:3674-3678. 9. Fauci, A. S., R. C. Gallo, S. Koenig, J. Salk, and R. H. Purcell. 1989. Development and evaluation of a vaccine for human immunodeficiency virus (HIV) infection. Ann. Intern. Med. 110:373-385. 10. Gnann, J. W., Jr., J. A. Nelson, and M. B. A. Oldstone. 1987. Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus. J. Virol. 61:2639-2641. 11. Gorny, M., J.-Y. Xu, V. Gianakakos, S. Karwowska, C. Williams, H. W. Sheppard, C. V. Hanson, and S. Zolla-Pazner. 1991. Production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type 1 envelope glycoprotein. Proc. Natl. Acad. Sci. USA 88:3238-3242. 12. Goudsmit, J., C. Debouck, R. H. Meloen, L. Smit, M. Bakker, D. M. Asher, A. V. Wolff, C. J. Gibbs, Jr., and D. C. Gajdusek. 1988. Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc. Natl. Acad. Sci. USA 85:4478-4482. 13. Ho, D. D., M. S. C. Fung, Y. Cao, X. L. Li, C. Sun, T. W. Chang, and N.-C. Sun. 1991. Another discontinuous epitope on glycoprotein gpl20 that is important in human immunodeficiency virus type 1 neutralization is identified by a monoclonal antibody. Proc. Natl. Acad. Sci. USA 88:8949-8952. 14. Ho, D. D., J. C. Kaplan, I. E. Rackauskas, and M. E. Gurney. 1988. Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization. Science 239:1021-1023.
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15. Ho, D. D., J. A. McKeating, X. L. Li, T. Moudgil, E. S. Daar, N.-C. Sun, and J. E. Robinson. 1991. Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody. J. Virol. 65:489-493. 16. Ho, D. D., M. G. Sarngadharan, M. S. Hirsch, R. T. Schooley, T. R. Rota, R. C. Kennedy, T. C. Chanh, and V. L. Sato. 1987. Human immunodeficiency virus neutralizing antibodies recognize several conserved domains on the envelope glycoproteins. J. Virol. 61:2024-2028. 17. Javaherian, K., A. J. Langlois, G. J. LaRosa, A. T. Profy, D. P. Bolognesi, W. C. Herlihy, S. D. Putney, and T. J. Matthews. 1990. Broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of HIV-1. Science 250:1590-1593. 18. Javaherian, K., A. J. Langlois, C. McDanal, K. L. Ross, L. I. Eckler, C. L. Jellis, A. T. Profy, J. R. Rusche, D. P. Bolognesi, S. D. Putney, and T. J. Matthews. 1989. Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Natl. Acad. Sci. USA 86:6768-6772. 19. Kenealy, W. R., T. J. Matthews, M.-C. Ganfield, A. J. Langlois, D. M. Waselefsky, and S. R. Petteway, Jr. 1989. Antibodies from human immunodeficiency virus-infected individuals bind to a short amino acid sequence that elicits neutralizing antibodies in animals. AIDS Res. Hum. Retroviruses 5:173-182. 20. Kennedy, R. C., G. R. Dreesman, T. C. Chanh, R. N. Boswell, J. S. Allan, T.-H. Lee, M. Essex, J. T. Sparrow, D. D. Ho, and P. Kanda. 1987. Use of a resin-bound synthetic peptide for identifying a neutralizing antigenic determinant associated with the human immunodeficiency virus envelope. J. Biol. Chem. 262:5769-5774. 21. Kennedy, R. C., R. D. Henkel, D. Pauletti, J. S. Allan, T. H. Lee, M. Essex, and G. R. Dreesman. 1986. Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein. Science 231:1556-1559. 22. LaRosa, G. J., J. P. Davide, K. Weinhold, J. A. Waterbury, A. T. Profy, J. A. Lewis, A. J. Langlois, G. R. Dreesman, R. N. Boswell, P. Shadduck, L. H. Holley, M. Karplus, D. P. Bolognesi, T. J. Matthews, E. A. Emini, and S. D. Putney. 1990. Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science 249:932-935. 23. Matsushita, S., M. Robert-Guroff, J. Rusche, A. Koito, T. Hattori, H. Hoshino, K. Jawaherian, K. Takatsuki, and S. Putney. 1988. Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope. J. Virol. 62:2107-2114. 24. McKeating, J. A., J. Gow, J. Goudsmit, L. H. Pearl, C. Mulder, and R. A. Weiss. 1989. Characterization of HIV-1 neutralization escape mutants. AIDS 3:777-784. 25. Meloen, R. H., R. M. Liskamp, and J. Goudsmit. 1989. Specificity and function of the individual amino acids of an important determinant of human immunodeficiency virus type 1 that induces neutralizing activity. J. Gen. Virol. 70:1505-1512. 26. Nara, P. L., and J. Goudsmit. 1990. Neutralization-resistant variants of HIV-1 escape via the hypervariable immunodominant V3 region: evidence for a conformational neutralization epitope, p. 297-306. In F. Brown, R. M. Chanock, H. S. Ginsberg, and R. A. Lerner (ed.), Vaccines 90. Modern approaches to new vaccines including prevention of AIDS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 27. Nara, P. L., L. Smit, N. Dunlop, W. Hatch, M. Merges, D. Waters, J. Kelliher, R. C. Gallo, P. J. Fischinger, and J. Goudsmit. 1990. Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees. J. Virol. 64:3779-3791. 28. Norley, S. G., and R. Kurth. 1991. Neutralizing antibodies and antigens in AIDS. Infection 19(Suppl. 2):S83-S88. 29. Palker, T. J., M. E. Clark, A. J. Langlois, T. J. Matthews, K. J. Weinhold, R. R. Randall, D. P. Bolognesi, and B. F. Haynes. 1988. Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides. Proc. Natl. Acad. Sci. USA 85:1932-1936.
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30. Papsidero, L. D., M. Sheu, and F. W. Ruscetti. 1989. Human immunodeficiency virus type 1-neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping. J. Virol. 63:267-272. 31. Prince, A. M., D. Pascual, L. B. Kosolapov, D. Kurokawa, L. Baker, and P. Rubinstein. 1987. Prevalence, clinical significance, and strain specificity of neutralizing antibody to the human immunodeficiency virus. J. Infect. Dis. 156:268-272. 32. Profy, A. T., P. A. Salinas, L. I. Eckler, N. M. Dunlop, P. L. Nara, and S. D. Putney. 1990. Epitopes recognized by the neutralizing antibodies of an HIV-1-infected individual. J. Immunol. 144:4641-4647. 33. Putney, S. D., K. Javaherian, J. Rusche, T. Matthews, and D. P. Bolognesi. 1990. Features of the HIV envelope and development of a subunit vaccine, p. 3-61. In S. D. Putney and D. P. Bolognesi (ed.), AIDS vaccine research and clinical trials. Marcel Dekker, New York. 34. Robertson, G. A., B. M. Kostek, W. A. Schleif, J. A. Lewis, and E. A. Emini. 1988. A microtiter cell-culture assay for the determination of anti-human immunodeficiency virus neutralizing antibody activity. J. Virol. Methods 20:195-202. 35. Rosner, B. 1990. Fundamentals of biostatistics. PWS-Kent Publishers, Boston. 36. Rossi, P., V. Moschese, P. A. Broliden, C. Fundaro, I. Quinti, A. Plebani, C. Giaquinto, P. A. Tovo, K. Ljunggren, J. Rosen, H. Wigzell, M. Jondal, and B. Wahren. 1989. Presence of maternal antibodies to human immunodeficiency virus 1 envelope glycoprotein gpl20 epitopes correlates with the uninfected status of children born to seropositive mothers. Proc. Natl. Acad. Sci. USA 86:8055-8058. 37. Rusche, J. R., K. Javaherian, C. McDanal, J. Petro, D. L. Lynn, R. Grimaila, A. Langlois, R. C. Gallo, L. 0. Arthur, P. J. Fischinger, D. P. Bolognesi, S. D. Putney, and T. J. Matthews. 1988. Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gpl20. Proc. Natl. Acad. Sci. USA 85:3198-3202. 38. Scott, C. F., Jr., S. Silver, A. T. Profy, S. D. Putney, A. Langlois, K. Weinhold, and J. E. Robinson. 1990. Human monoclonal antibody that recognizes the V3 region of human immunodeficiency virus gpl20 and neutralizes the human T-lymphotropic virus type III MN strain. Proc. Natl. Acad. Sci. USA 87:85978601. 39. Skinner, M. A., A. J. Langlois, C. B. McDanal, J. S. McDougal, D. P. Bolognesi, and T. J. Matthews. 1988. Neutralizing antibodies to an immunodominant envelope sequence do not prevent gpl20 binding to CD4. J. Virol. 62:4195-4200. 40. Starcich, B. R., B. H. Hahn, G. M. Shaw, P. D. McNeely, S. Modrow, H. Wolf, E. S. Parks, W. P. Parks, S. F. Josephs, R. C. Gallo, and F. Wong-Staal. 1986. Identification and characterization of conserved and variable regions in the envelope gene of HTLV-III/LAV, the retrovirus of AIDS. Cell 45:637-648. 41. Thali, M., U. Olshevsky, C. Furman, D. Gabuzda, M. Posner, and J. Sodroski. 1991. Characterization of a discontinuous human immunodeficiency virus type 1 gpl20 epitope recognized by a broadly reactive neutralizing human monoclonal antibody. J. Virol. 65:6188-6193. 42. Wain-Hobson, S., J.-P. Vartanian, M. Henry, N. Chenciner, R. Cheynier, S. Delassus, L. P. Martins, M. Sala, M.-T. Nugeyre, D. Guetard, D. Klatzmann, J.-C. Gluckman, W. Rozenbaum, F. Barre-Sinoussi, and L. Montagnier. 1991. LAV revisited: origins of the early HIV-1 isolates from Institut Pasteur. Science 252:961-965. 43. Warren, R. Q., H. Wolf, K. R. Shuler, J. W. Eichberg4 R. A. Zajac, R. N. Boswell, P. Kanda, and R. C. Kennedy. 1990. Synthetic peptides define the fine specificity of the human immunodeficiency virus (HIV) gpl60 humoral immune response in HIV type 1-infected chimpanzees. J. Virol. 64:486-492. 44. Zwart, G., H. LanfedUk, L. van der Hoek, J.-J. de Jon, T. F. W. Wolfs, C. Ramautarsing, M. Bakker, A. de Ronde, and J. Goudsmit. 1991. Immunodominance and antigenic variation of the principal neutralization domain of HIV-1. Virology 181:481489.
JOURNAL OF VIROLOGY, Sept. 1992, p. 5210-5215
0022-538X/92/095210-06$02.00/0 Copyright © 1992, American Society for Microbiology
Examination of Sera from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals for Antibodies Reactive with Peptides Corresponding to the Principal Neutralizing Determinant of HIV-1 gpl20 and for In Vitro Neutralizing Activity RONALD Q. WARREN,1'2 STEPHANIE A. ANDERSON,1'2 WATOKY M. M. M. NKYA,3 JOHN F. SHAO,4 CRAIG W. HENDRIX,2'5 GREGORY P. MELCHER,2'5 ROBERT R. REDFIELD,6 AND RONALD C. KENNEDY' 2* Department of Virology and Immunology1 and Center for AIDS Research,2 Southwest Foundation for Biomedical Research, San Antonio, Te-xas 78284-0147; Kilimanjaro Christian Medical Center, Moshi, 3 and Muhimbili Medical Center, Dar es Salaam, 4 Tanzania; Department of Medicine, Wilford Hall Medical Center, Lackland Air Force Base, Texas 782365; and Department of Retroviral Research, Walter Reed Army Institute of Research, Rockville, Maryland 208506 Received 10 January 1992/Accepted 1 June 1992
Sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals from the United States and Tanzania were examined for antibody reactivity to four synthetic peptides which corresponded to the principal neutralizing determinant from the V3 region of HlV-1 gpl20. We observed that the majority of sera from both countries contained antibodies reactive with a V3 peptide whose sequence is based on that of the HIV-1 MN isolate. We were unable to establish a relationship between the presence of V3-reactive antibodies, as measured by enzyme-linked immunosorbent assay and neutralization of homologous HIV-1 isolates, in sera from either the United States or Tanzania. We observed that some sera which contained high antibody titers to the V3 peptides failed to neutralize HIV-1, while others with no antibody reactivity to the panel of V3 peptides exhibited in vitro neutralizing activity. These results suggest that neutralizing epitopes exist outside the V3 loop and that the presence of V3-reactive antibodies in sera does not imply in vitro neutralization of the homologous HIV-1 isolate. In addition, it appears that the V3 loop may consist of both neutralizing and nonneutralizing epitopes. The identification of neutralizing as well as nonneutralizing epitopes will be important for the design of potential HIV-1 vaccines. The principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) is thought to consist of a disulfide-linked loop structure spanning the third variable region (V3) of gpl20 (18). This immunodominant epitope corresponds to amino acid residues 303 to 338 of gpl20 (IIIB) (40) and has been reported to induce type-specific neutralizing antibodies in vitro (11, 12, 22, 37, 38). On the basis of the induction of neutralizing antibodies in experimental animals, this viral epitope is under investigation as a candidate for potential HIV-1 vaccines and as a target for immunoprophylactic strategies (8, 9, 33). Thus, characterization of the immune response to this epitope following natural infection
from infected individuals by enzyme-linked immunosorbent assay (ELISA) and their ability to neutralize homologous HIV-1 isolates in vitro. This lack of correlation between antibodies to V3 epitopes defined by synthetic peptides and neutralizing activity suggests that conformational epitopes associated with V3 or non-V3 epitopes may play an important role in the neutralization of HIV-1 among infected individuals.
MATERIALS AND METHODS Human sera. Serum specimens from HIV-1-infected individuals from the United States (n = 37) and Tanzania (n = 74) were examined in this study. All specimens from HIV1-infected individuals were confirmed to contain anti-HIV-1 gpl60 antibodies by Western immunoblot analysis (Bio-Rad Laboratories, Richmond, Calif.). Serum specimens from the United States were obtained from U.S. military personnel. Because of the limitations in the quantity of sera available from HIV-1-infected individuals from Tanzania, 15 serum samples were selected to be characterized in the neutralization assays. Ten serum samples from healthy, uninfected individuals were used as controls. All serum samples were heat inactivated at 56°C for 30 min prior to use. Synthetic V3 region peptides. Peptides RP135, RP142, and RP145, which correspond to the gpl20 V3 region of HIV-1
with HIV-1 in humans from geographically diverse populations is important for the design and effectiveness of these potential strategies for controlling HIV-1 infection. In this study, we examined sera from HIV-1-infected individuals from both the United States and Tanzania for antibodies reactive with four synthetic peptides whose sequences are based on the V3 region. Specifically, we compared sera for antibody reactivity to the V3 peptides together with their ability to neutralize HIV-1 isolates in vitro. The results described herein indicate that there is little correlation between the detection of V3-reactive antibodies in sera *
Corresponding author. 5210
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ANTIBODY REACTIVITY TO V3 PEPTIDES
TABLE 1. Amino acid sequences of principal neutralizing determinant-derived V3 synthetic peptides used in this study
Peptide
(amino acid residues) 304-321 RP135 (308-331) RP142 (306-330) RP145 (303-325)
HIV-1
Amino acid
isolate
IIIB IIIB
sequence0
(CGY)TRPNNNTRKSIRIQRGPG NNTRKSIRIQRGPGRAFVTIGKIG(C)
MN
YNnKBRIHIGPGRAFYTTKNIIG(C)
HAN/SC
NNTRKGIHIGPGRA/FYATGDIIG(C)b
a Amino acids in parentheses were added to facilitate coupling to carrier proteins for related immunization studies. b The slash mark indicates division between HAN and SC amino acid sequences.
isolates IIIB (LAI) (42) and MN and the composite HAN/ SC, respectively, were kindly supplied by Scott Putney (Repligen, Cambridge, Mass.). The composite V3 peptide, RP145, was examined to assess whether hybrid sequences could be recognized by antibodies from naturally infected individuals (17). Peptide 304-321, corresponding to the IIIB isolate, was synthesized by methods previously described (43). The amino acid sequences of the four V3 region-derived peptides are depicted in Table 1. Determination of antibody binding to synthetic peptides as measured by ELISA. The methods for detecting antibody reactivity to HIV-1 gpl60 synthetic peptides by ELISA have been described in detail previously (5, 21, 43). All antisera (1/50 dilution) were screened for V3-reactive antibodies in at least three separate determinations. Ten HIV-seronegative normal control sera were similarly tested for antibodies reactive to the synthetic peptides. Experimental optical density (OD) values greater than three times the mean OD value obtained for the 10 control sera were considered positive in reactivity for a given peptide. This positive reactivity cutoff level for these sera exceeded the 95% confidence level. In those instances when normal control sera resulted in a negligible OD value and a cutoff value for positive reactivity could not be calculated, an OD value of 0.1 was used as the positive cutoff. Endpoint antibody titers of sera were determined to be the greatest dilution of sera which resulted in an OD value greater than the positive cutoff. Neutralization assays. Two neutralization assays were performed in this study. In the first assay, sera to be examined were preincubated for 1 h with 100 50% tissue culture infective doses of HIV-1 isolate IIIB. This mixture (50 ,ul) was then added to microtiter wells containing 25,000 SupTl cells (100 ,ul, final volume). Cells were incubated at 37°C for
5211
8 days, being fed on days 3 and 6 with RPMI containing 15% fetal bovine serum. On day 8, 10 ,ul of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; thiazolyl blue; 0.012 M; Sigma, St. Louis, Mo.) was added to the individual wells for 4 h at 37°C. The cultures were then inactivated by the addition of 175 ,ul of 0.04 N HCI in isopropanol, and the OD at 570 nm was determined. Endpoint neutralization titers were calculated as the greatest dilution of sera which resulted in 50% target cell viability in comparison with control wells containing normal sera (34). The MTT neutralization assay was performed only with the IIIB isolate, since the MN isolate was not cytotoxic toward the SupTl target cells. In the second assay, a p24 antigen capture ELISA was used to determine neutralizing activity of sera against both the IIIB and MN isolates (100 50% tissue culture infective doses) (Retrovirology Coulter Corp., Hialeah, Fla.). The cells and virus were cultured as described above, with the HIV-1 p24 levels in culture supematant being determined on day 8 according to the manufacturer's recommended procedure. Sera were considered to contain neutralizing activity if greater than 50% reductions in p24 levels were detected in comparison with wells containing normal sera at a 1/50 dilution. Neutralization endpoint titers were determined as described above. Because of the variation in target cell susceptibility to infection by various HIV-1 isolates, we also examined CEM-SS cells as target cells in the neutralization assays. The p24 ELISA was used to assess neutralization of both the IIIB and MN isolates with the CEM-SS target cell line. Briefly, 50,000 CEM-SS target cells were adhered onto a poly-L-lysine-coated microtiter plate. Following a 1-h incubation at 25°C, 50 ,ul of the virus-serum mixture was added to the target cells, and the mixture was allowed to incubate for 8 days at 37°C. Supernatants were harvested and screened for p24 reactivity as described above. Statistical analysis. Sera were examined for the significance of peptide-reactive antibodies and neutralizing activity by the chi-square test (35). In addition, correlation coefficients (r values) were calculated to compare antibody titers, as assessed by ELISA, and in vitro neutralization titers.
RESULTS
Serum specimens from HIV-1-infected individuals from the United States and Tanzania were examined for antibody reactivity to four V3-based synthetic peptides. These specimens were also examined for neutralizing activity against HIV-1 isolates IIIB and MN (Table 2). Peptide 304-321, which corresponds to the amino half of the V3 loop of the
TABLE 2. Summary of serum reactivity from HIV-1-infected individuals to peptides corresponding to the V3 region of gpl20 and neutralizing activity No. (%) of sera with neutralizing No. (%) ofagainst of sera reactive to V3 peptide: HIV-1 isolate: activity serumN. (%
Source of serum
Source of
samples
IIIB 304-321
RP135
RP142
MN
RP145 p24
MIT
p24
5 (14) 10 (27) 31 (84)a 19 (51) 4 (11) 21 (57) United States (n = 37) 6 (16) 14 (19) 15 (20) 48 (65)39 (53) Tanzania (n = 74) 7 (47) 3b (27) 11C (73) a < Represents a statistically greater percentage of serum reactivity to RP142 than to RP135 (P 0.001). b Eleven representative serum samples from Tanzania were examined for neutralizing activity by p24 activity against the IIIB isolate. c Fifteen representative serum samples from Tanzania were examined for neutralizing activity by MTT uptake against the IIIB isolate and by p24 activity
against the MN isolate.
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WARREN ET AL.
IIIB isolate, was reactive with 5 of 37 serum specimens (14%) from the United States and 14 of 74 specimens (19%) from Tanzania by ELISA. Peptide RP135, which corresponds to the central portion of the V3 loop of the IIIB isolate, was reactive with a higher percentage of U.S. specimens (10 of 37 [27%]) than was peptide 304-321. Similar percentages of HIV-1-infected specimens from Tanzania were reactive with peptides RP135 (20%) and 304-321 (19%). Peptides RP142 and RP145, which correspond to the MN and HAN/SC isolates, respectively, were reactive with the majority of serum specimens examined from both countries. We observed that 31 of 37 serum specimens (84%) from the United States and 48 of 74 specimens (65%) from Tanzania were reactive with RP142. Nearly equal percentages of specimens from both countries were reactive with RP145 (51% from the United States and 53% from Tanzania). A statistically greater percentage of serum specimens from both the United States and Tanzania contained antibodies reactive with RP142 than with RP135 (P > 0.001), suggesting the widespread prevalence of HIV-1 MN-like isolates in both countries. Next, we examined these same serum specimens for their ability to neutralize HIV-1 isolates IIIB and MN in vitro and compared this activity with peptide ELISA reactivity. Two separate assays were used to determine neutralizing activity of sera against the IIIB isolate. These two assays measure different aspects of viral neutralization, since the p24 capture ELISA indicates the level of viral replication, while the MTT uptake assay measures the amount of target cell cytotoxicity by HIV-1. As seen in Table 2, a high percentage of serum specimens from both countries neutralized the IIIB isolate when assessed by the cellular cytotoxicity assay. We observed that 21 of 37 serum specimens (57%) from the United States and 11 of 15 serum specimens (73%) from Tanzania contained neutralizing activity. In contrast, fewer specimens exhibited neutralizing activity against IIIB when levels of p24 were measured in the culture supernatants. It therefore appears that relatively low levels of virus may lead to elevated p24 levels and no in vitro neutralizing activity as assessed by this method. However, under these conditions, target cell viability can remain high in vitro, indicating that a given serum sample may exhibit neutralizing activity, as judged from inhibition of HIV-i-induced cytotoxicity. These results indicate that care must be taken in interpreting serum neutralizing activity according to which assay is used. Individual serum samples from the United States were further characterized for antibody reactivity to the panel of V3 peptides and examined for levels of neutralizing activity against IIIB and MN (Table 3). These samples were examined to determine the importance of V3-reactive antibodies in sera and their effect on neutralizing activity against homologous HIV-1 isolates in vitro. Again, serum neutralizing activity against the IIIB isolate was measured by both the p24 antigen capture ELISA and MTT uptake. We were unable to establish a correlation between the presence of V3 peptide-reactive antibodies in sera from these individuals and viral neutralizing activity by using either neutralization assay. For example, we found no significant correlation between antibody reactivity to the IIIB-based V3 peptide RP135 and neutralization of IIIB on the basis of reduction of HIV-1 p24 (P = 0.617) or MTT uptake (P 0.104). In addition, of the 26 serum samples which lacked antibodies to either IIIB-based peptide (304-321 or RP135), 17 exhibited neutralizing activity (MTT) against the IIIB isolate in vitro. We also observed that of the 31 serum samples containing =
TABLE 3. Comparison of antipeptide reactivity and in vitro HIV-1 neutralizing activity of sera from infected individuals from the United States Neutralization titer
Antibody reactivity Serumody Serum
reactlvlty
against HIV-1 IIIB
sample 304-321
RP135
RP142
RP145
p24
+ 1 Negb + + 2 Neg + + Neg 3 + Neg 4 + 5 Neg + + + 6 Neg + + 100 + 7 8 Neg + + 50 9 + 200 10 + 11 Neg + + + 12 Neg + + + + 13 Neg + 14 Neg Neg 15 + 16 Neg + + + 17 Neg + + + + 18 Neg + + + + 19 Neg + + 50 20 21 Neg + + + 22 Neg + + + 23 Neg + + 24 Neg 25 Neg + 26 Neg + 27 Neg + + 28 Neg + 29 Neg + 30 Neg + 31 Neg + 32 Neg + + Neg 33 + + + 34 Neg + + Neg 35 + 36 Neg + + 37 Neg a Reciprocal dilution of serum that either reduced p24
isolatesa: MN
MiTT
(p24) ('4
Neg
Neg
50 Neg 50 100
Neg 100 100
Neg 800 100 50 200
50 Neg Neg 400 Neg 200 Neg 100 400 50 800 Neg Neg 200 Neg Neg Neg Neg 50 Neg 50 50 Neg Neg 50 50 200
100
Neg Neg Neg Neg
Neg Neg 200
Neg 200
Neg Neg Neg Neg Neg > 1,600 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg
levels by 50% or resulted in 50% target cell viability. b Neg, no neutralizing activity was detected at a 1/50 dilution of serum.
antibodies to peptide RP142, only 6 neutralized the homologous MN isolate in vitro. A similar pattern of neutralization was observed with 15 selected serum samples from HIV-1-infected individuals from Tanzania, as was previously seen with U.S. samples (Table 4). Again we found that a higher percentage of samples neutralized the IIIB isolate (11 of 15 [73%]), as measured by MTT uptake, than contained antibodies reactive with either of the IIIB-based peptides 304-321 (0 of 15) and RP135 (5 of 15 [33%]). In addition, we observed that six of these samples neutralized the IIIB isolate by the MTT assay yet lacked antibody reactivity to either of the IIIBbased V3 peptides. More importantly, we found that a high percentage of Tanzanian serum samples contained antibodies reactive with RP142 (13 of 15 [87%]), while only 7 (47%) neutralized the MN isolate in vitro (P = 0.509). As with the U.S. samples, we observed no significant correlation between the presence of V3-reactive antibodies and neutraliz-
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ANTIBODY REACTIVITY TO V3 PEPTIDES
TABLE 4. Comparison of antipeptide reactivity and in vitro neutralizing activity of sera from HIV-1-infected individuals from Tanzania
Neutralization titer
Antibody reactivity to V3 peptide: Serum sample
IIIB 304-321
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
RP135
-
+ -
-
-
-
+ + + +
-
-
-
-
RP142
+ + + + + + + + + + + + +
RP145
+ + + + + + + + + + + + +
MN
p24
MIT
p24
NDa ND 50 Negb 50 Neg Neg Neg ND Neg Neg Neg Neg 200 ND
800 800 100 800 200 400 1,600 800 400 Neg 400
100 200 50 Neg 50 Neg Neg Neg > 1,600 Neg
Neg Neg Neg 400
Neg Neg Neg 200 > 1,600
ND, because of the limited availability of some serum samples from Tanzania, the neutralizing activity as assessed by this assay was not determined. b Neg, no neutralizing activity was detected at a 1/50 dilution of serum. a
ing activity in sera from HIV-1-infected individuals from Tanzania. Since HIV-1 isolates can vary significantly in their target cell tropism, we examined 10 serum samples from the United States for neutralizing activity, using two target cell lines, SupTl and CEM-SS. Similar neutralization titers were observed when sera were tested against either the SupTl or CEM-SS target cells (data not shown). Thus, low levels of serum neutralizing activity toward the MN isolate, compared with high levels of reactivity to RP142, were not influenced by the target cells used in the neutralization assays. We then examined 14 selected serum samples from both countries to determine whether antibody titers to the V3 region peptides would correlate with neutralizing titers against homologous HIV-1 isolates. We were interested in determining whether high antibody titers to RP142 would lead to similarly high levels of neutralizing antibodies against the MN isolate. As seen in Fig. 1, no significant correlation was
56004800 t 4000
t
aN
3200w
Q-
2400
t
1600 '
t
8001 0
50
100
150
200
250
Neutrolization Titer (MN)
FIG. 1. Correlation between antibody endpoint titers to RP142 in sera from HIV-1-infected individuals and neutralizing titers against HIV-1 MN in vitro.
5213
observed between antibody titers to RP142 and neutralization titers of MN in vitro (r = 0.086). Similar results were observed with sera containing antibodies to RP135 and neutralization titers against the IIIB isolate (r = 0.138) (data not shown). DISCUSSION The V3 loop region of gp120 represents an immunodominant site on the envelope of HIV-1 which elicits type-specific neutralizing antibodies in immunized animals (12, 16, 19, 22, 29, 32, 33, 36, 38, 39). Deletion of the V3 loop region from gpl60 has been reported to result in a truncated glycoprotein that is unable to stimulate neutralizing antibodies in immunized animals (18). Previous studies have suggested that the majority of HIV-1 neutralizing antibodies are directed against the V3 loop. For example, preincubation of peptides RP135 or RP142 with HIV-1 antisera reportedly removes all neutralizing antibodies (18, 37). Amino acid substitutions within the V3 region of HIV-1 have been reported to lead to the emergence of neutralization-resistant variants (24-27). In addition, several studies have shown that the neutralizing activity of sera from HIV-1-infected individuals can be blocked by preincubation with V3 peptides (8, 44). However, additional HIV-1 neutralizing epitopes lying outside the V3 region of gpl20 have also been identified (1, 10, 14, 16, 20, 28, 30, 41). It is therefore important to understand the relationship between the detection of anti-V3 antibodies in sera from naturally infected individuals and the neutralization of HIV-1 in vitro. In this study, we were unable to establish a correlation between the presence of V3-reactive antibodies in sera and the neutralization of homologous HIV-1 isolates in vitro. For example, of 31 serum samples from the United States which contained antibodies reactive with RP142, only 6 neutralized the homologous MN isolate in vitro. We also found little correlation between the presence of antibodies to peptide 304-321 or RP135 and the ability of sera to neutralize IIIB. Carrow and colleagues reported a similar lack of correlation between the presence of anti-V3 antibodies in sera and neutralization of the IIIB isolate (4). However, these investigators did observe a significant correlation between the presence of anti-V3 antibodies and neutralization of the MN isolate. Our results also differ from those of Zwart and colleagues, who described a correlation between a single human serum sample containing V3-reactive antibodies and neutralizing activity in vitro (44). By using two separate neutralization assays, we observed quite different levels of serum neutralizing activity against the IIIB isolate. These differences can be attributed to the fact that the p24 antigen capture ELISA reflects the level of virus in the culture supernatant, while the MTT dye uptake assay measures the degree of cellular cytotoxicity in vitro. Our data indicate that while certain sera may protect target cells against HIV-1-mediated cytotoxicity, low levels of viable HIV-1 may remain in these cultures, with no neutralizing activity being observed when assessed by the HIV-1 p24 assay. Alternatively, the assay based on inhibition of cellular cytotoxicity may be more sensitive than the assay for reduction of HIV-1 p24. We observed that a high percentage of serum samples from both the United States and Tanzania contained antibodies reactive with the RP142 peptide. These results agree with those of earlier studies suggesting that the MN isolate, or antigenically related isolates, is commonly found worldwide (4, 6, 22, 44). In contrast, two peptides whose sequences correspond to that of the IIIB isolate (304-321 and
5214
WARREN ET AL.
RP135) were reactive with fewer than one-third of serum samples from either the United States or Tanzania. This low level of serum reactivity agrees with previous reports suggesting that the IIIB isolate currently represents a minor population of HIV-1 worldwide (6). Peptide RP135 appears to express additional epitopes in comparison with peptide 304-321, since a higher percentage of HIV-1-infected serum samples from the United States reacted with RP135. Peptide RP145, which corresponds to a composite of two HIV-1 isolates (HAN and SC), was reactive with approximately one-half of the Tanzanian and U.S. serum samples tested. We also examined whether serum endpoint titers to the V3 peptides would influence neutralization titers against homologous HIV-1 isolates. We were unable to establish a significant correlation between antibody titers to V3 peptides by ELISA and neutralizing activity. The presence of high antibody titers to RP142 (>1,600) had little effect on whether individual sera would neutralize the MN isolate in vitro. A similar lack of correlation between antibody titers to HIV-1 and neutralizing activity in vitro has been previously reported (31). Together, these results suggest that neutralizing epitopes lying outside the V3 region of gpl20 may play an important role in HIV-1 neutralization in vitro. The importance of target cells on serum neutralizing activity was also examined. Neutralization titers of sera appeared similar when two target cell lines were used (data not shown). Few sera neutralized the MN isolate when either the SupTl or CEM-SS cells were used as targets. These results further indicate that while most sera contained RP142-reactive antibodies by ELISA, few demonstrated neutralizing activity against the MN isolate in vitro. The observation of sera which contain V3-reactive antibodies yet lack neutralizing activity suggests that nonneutralizing epitopes may reside within the V3 loop. Antibodies reactive with these epitopes may demonstrate V3 reactivity by ELISA while exhibiting no neutralizing activity in vitro. A study by D'Souza and colleagues, using monoclonal antibodies specific for V3 epitopes, similarly found that antibody reactivity to V3 peptides did not equate with neutralizing activity (7). The amino acid sequence GPGR has been identified as an important target for in vitro neutralizing antibodies (2, 3, 23). Our study indicates that the majority of HIV-1-infected individuals do not produce antibodies reactive with this particular V3 epitope. The data presented here agree with those of previous studies suggesting that conformational V3 epitopes, along with epitopes lying outside the V3 region, play important roles in the neutralization of HIV-1 (13, 15). Our study indicates that the presence of antibodies reactive with V3-based synthetic peptides does not imply that sera will neutralize homologous HIV-1 isolates in vitro. In addition, we identified sera which lacked detectable anti-V3 antibodies by ELISA yet neutralized homologous HIV-1 isolates at high titers. Neutralizing sera which lack V3-reactive antibodies have been described recently by other investigators (2). It therefore appears that neutralizing epitopes which lie within the V3 region, along with those which lie outside it, will play equally important roles in potential vaccine development. In addition, our data suggest that the fine specificity of the humoral immune response to the V3 region may differ between experimentally immunized animals and naturally infected humans. Since other investigators have demonstrated that animals immunized with either RP135 or RP142 produce type-specific neutralizing antibodies, it appears that the V3 loop may contain neutralizing as well as nonneutralizing epitopes (18).
J. VIROL.
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