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Broadly neutralizing, MN-like PND-directed antibodies in rwandan children with long-term HIV1 infection
© INSTrrUT PASTEUR/ELsEVIER Paris 1995
Res. Virol. 1995, 146, 201-210
Broadly neutralizing, MN-like PND-directed antibodies in Rwandan children with long-term HIV1 infection C. Fraisier (~) (*), P. Van de Perre (2), p. Lepage (3), D.G. Hitimana (3), E. Karita (2) and C. Desgranges (~) (**~ (I) INSERM U271, 151 Cours A. Thomas, 69424 Lyon Cedex 03 (France), (2) National A1DS Control Programme Ministry of Health, Kigali (Rwanda), and (~) Centre Hospitalier de Kigali, Kigali (Rwanda)
SUMMARY
Sera from 11 perinatally HIVl-infected Rwandan children with prolonged survival were tested in vitro for the presence of neutralizing antibodies against different HIV1 strains. These 11 sera from long survivor (LS) children were compared with 16 sera from Rwandan children with AIDS. Sera from HIVl-infected children exhibited the greatest neutralizing activity against HIVIMN cell-free infection. They also inhibited HIVIRu and HIVIL~ cell-free infection with lower titres. Higher neutralization titres were observed in sera-from LS compared to the AIDS group, with a significant difference for HIVIMN and HIVIL~J strains. Sera from LS children also inhibited syncytium formation induced by HIVIMN-infected cells with higher titres than AIDS children. Sera from the HIVl-infected children showed reactivity to the HIVIMN V3 peptide, as well as to both the US/European and the African consensus V3 peptides. Higher reactivity was observed in sera from LS than from AIDS children, and the difference was significant toward the African consensus peptide. The LS children also had significantly higher V3MN IgG avidity than the AIDS children. These data support the notion that the humoral response to the V3 domain, associated with a broadly neutralizing activity, may be an important factor in the prolonged survival of these children. The specificity against HIVIMN also suggests that an antigenically MN-related strain may be prevalent in Rwanda, and that an MN-related principal neutralizing domain sequence could be an important determinant for candidate vaccines in this part of Africa. Key-words: HIV1, AIDS, Child, IgG; Rwanda, Perinatal infection, Long-term survival, HlVlMN, V3 loop, Neutralizing antibodies, IgG avidity, Vaccination strategies.
INTRODUCTION Infection with human immunodeficiency virus type 1 (HIV1) leads to profound, progressive impairment of T-cell function and to the develop-
ment of the acquired i m m u n o d e f i c i e n c y syndrome (AIDS) (Bowen et al., 1985). HIV1 infection has been shown to progress more rapidly in children than in adults (Fallon et al., 1989; Oxtoby, 1990). H o w e v e r , p e r i n a t a l l y HIV1-
Submitted November 11, 1994, accepted January 25, 1995. (*) Presentaddress: Lab. of Gene Structureand Expression,NationalInstitutefor Medical Research,The Ridgeway,Mill Hill, London NW71AA,UK. (**) Correspondingauthor.
C. FRAISIER E T AL.
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infected children surviving to over 5 years of age have recently been reported (Blanche et al., 1990; Lepage et al., 1991; Tovo et al., 1992). We described the prolonged course of HIV 1 infection in 16 African children maternally infected by HIV1 (Lepage et al., 1991). Most of the children who survived beyond 5 years presented with moderate signs and symptoms. Numerous biological markers have been associated with long-term survival, such as low viral load, the presence of non-syncytium-inducing and monocytotropic HIV1 strains and HIVl-specific CD8 + T-cell immune responses (Levy, 1993). In addition to the cellular response, neutralizing antibodies directed against HIV1 envelope glycoproteins could be an important component of the protective immune response. We previously reported that prolonged survival was associated with persistent production of IgM to HIV 1 envelope proteins and of IgG to p17 (Van de P e r r e e t al., 1992). Antibodies from HIVl-infected individuals could neutralize a broad variety of HIV1 isolates. These neutralizing antibodies, directed against conformational epitopes of g p l 2 0 , block the gpl20 interaction with the CD4 receptor (Steimer et al., 1991), and a decline in antibodies to the CD4-binding site has been correlated with rapid disease progression (Cavacini et al., 1993). Furthermore, four major neutralizing regions of HIV1 glycoproteins have been identified (Broliden et al., 1992), and a n t i b o d i e s against restricted epitopes of gpl20 and gp41 have been shown to be significantly less prevalent in AIDS than in asymptomatic patients (Neurath et al., 1990). In particular, a correlation has been demonstrated between the level of antibodies to the aa303-338 region and development of the disease (Neurath et al., 1990). This region, located in the third hypervariable region of gpl20 (V3), forms a loop between two conserved cysteines at positions 303 and 338, and has been determined to be the principal neutralizing domain (PND) of HIV1
LS mAb NBCS PBS-T
= = = =
l o n g survivor. monoclonal antibody. new-born calf serum. p h o p h a t e - b u f f e r e d saline + T w e e n - 2 0 .
(Javaherian et al., 1989). The PND has also been identified as a fusion domain (Freed et al., 1991) and is essential for gp 120-mediated syncytia formation (Travis et al., 1992). It also contains target-cell-specific determinants of membrane fusion (Bergeron et al., 1992) and tropism for T cells and macrophages (Chesebro et al., 1992; Hwang et al., 1991). The V3 loop was also related to both syncytium-inducing and replicating phenotypes of HIV1 (De Jong et al., 1992; F o u c h i e r et al., 1992). Antibodies directed against linear epitopes of the V3 loop are generally defined as type-specific and react with individual viral isolates. However, the neutralization of diverse HIV1 isolates could be observed with monoclonal antibodies (mAb) directed against the V3 domain. These broadly neutralizing antibodies were, in particular, obtained by immunization with HIVIMN V3 peptides (Ohno et al., 1991 ; White-Scharf et al., 1993) or defined by their reactivity against the HIV1MN V3 loop (Gorny et al., 1992). These anti-V3 antibodies are able to block infectivity in vitro, and it has also been shown that g p l 2 0 V 3 - d o m a i n - s p e c i f i c mAbs could prevent HIV 1 infection in chimpanzees (Emini et al., 1992) as well as in hu-PBLSCID mice (Safrit et aL, 1993). The results presented here reveal the presence of broadly neutralizing activity and MN-like PND-specific antibodies with higher V3MN IgG avidity in sera from long-term survivor (LS) children. This activity could be involved in the less severe course of HIV1 disease in these children.
PATIENTS
AND
METHODS
Patients
A group of 16 out of 537 children born to HIV1infected mothers and more than 5 years old at enrollment were diagnosed at the Paediatric Department of the Centre Hospitalier de Kigali, Kigali, Rwanda.
PND RT Sn
= = =
principal neutralizing domain. room temperature. seronegative.
ANTIBODIES IN RWANDAN
CHILDREN WITH LONG-TERM HIV1 INFECTION
Sera from 11 out of these 16 children (LS) were collected in February and March 1990. These sera were compared in our study with sera from 16 Rwandan children 18 months to 6 years old who fulfilled the World Health Organization definition of paediatric AIDS (WHO, 1986) (AIDS) and 4 sera from H I V I seronegative (Sn) Rwandan children 6 to 7 years old.
Virus stock Virus suspensions of HIV1 strains were obtained f r o m supernatants of HIV1MN ( N o r t h - A m e r i c a ) , HIV1LM (Europe) and HIV1Ru (Zaire) chronically infected H9 cells provided by the National Institutes of Health (NIH), passed through 0.45-Bm filters and stored at - 8 0 ° C before use. For each virus stock, a MT4-cell infectivity endpoint was defined as the last dilution of the virus stock that killed 100% MT4 cells in a 7-day assay (Gorny et al., 1992; Robertson et al., 1988).
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tide at 1 p.g/ml in 0.1 M carbonate buffer pH 9.5. Plates w e r e w a s h e d t w i c e with PBS c o n t a i n i n g 0.1% Tween-20 (PBS-T), saturated for 30 min at 37°C with 200 ~1 o f PBS + 10% n e w - b o r n c a l f serum (NBCS) and incubated for 1 h at room temperature (RT) with 100 ~tl of sera at 1:100 dilution in 5 x P B S - T - 1 0 % NBCS. After extensive washing, bound antibodies were detected with peroxidaseconjugated goat anti-human immunoglobulins for 1 h at RT. Then, the enzymatic reaction was developed with the substrate, O-phenylenediamine, and read at 492 nm with a "Titertek Multiscan" (Flow Laboratories, Irvine, Scotland, UK). Cut-off values were set at 3 SD plus mean absorbance of the four negative controls. For the V3MN IgG avidity measurement, 100 gtl of the serum dilution giving an OD of 1.0 +_ 0.1 was d i s s o c i a t e d f r o m the V3MN solid p h a s e at 100 ng/well with 100 I11 of 8 M urea. The avidity index was calculated as (OD with 8 M urea/OD without urea) × 100.
Cell-to-cell syncytium formation inhibition assay Synthetic peptides Peptides corresponding to the g p l 2 0 PND were kindly p r o v i d e d by the A g e n c e N a t i o n a l e de la Recherche sur le S I D A and were synthesized by Neosystem Laboratories (Strasbourg, France). Consensus V3 peptides were kindly provided by Dr F. Barin and Dr A. Baillou (URA CNRS 1334, Tours, France). The peptide referred to as V3Cons (table I) c o r r e s p o n d e d to the U S / E u r o p e a n c o n s e n s u s sequence determined by Larosa et al. (1990). The peptide referred to as V3AfCons2 corresponded to the African consensus sequence derived from the V3 sequences of 108 African isolates (Myers et al., 1991).
Serial 2-fold dilutions (starting at 1:20) of heatinactivated sera (100 ~tl) were incubated in a microplate with 50 ~tl of HIV1LM- or HIV1MN-infected H9 cells at 10~cells/ml for 1 h at 37°C, 5 % CO 2. Fifty microliters of non-infected SupT1 target cells at 2 × 105 cells/ml were then added. After 24 h of culture at 37°C, syncytia were enumerated by light microscopy. In the control wells without serum, 50 to 80 multinucleated giant cells were observed. The neutralization titre was defined as the reciprocal of the highest dilution resulting in 5 0 % reduction in the number of syncytia when compared to the control wells without serum.
Neutralization of the cytopathic effect ELISA Microtitre plates (Immulon 2, Dynatech Laboratories Inc, Chantilly, VA, USA) were coated overnight at 37°C with 100 B1 of the V3 synthetic pep-
This assay was performed as described elsewhere ( R o b e r t s o n et al., 1988). Serial 2-fold dilutions (starting at 1:20) of heat-inactivated sera (50 pJ) were incubated for 1 h at r o o m temperature with
Table I. V3 loop sequences. MN V3Cons LAI RF Z2 V3AfCons2
CNKRKRIHI G NTRKSIHI G CNTRKS I R I QRG CNTRKS I TKG CNIRQRTPI G NTRQRTRI G
PGRAFYTT PGRAFYPGR AFVPGRVI YLGQSLYPGQAFY-
KN T TGE I IGDI T IGK A TGQ T TKTRS T TGI I GD
RQAHC
I RQAHC
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C. FRAISIER E T AL.
50 pl of HIV1MN, HIV1LAI or HIV1Rn viral supernatants at the last dilution giving 100% cell killing (endpoint dilution). MT4 cells (5 × 10 4 cells in 100 ~tl) were added. After a 7-day incubation at 37°C, cell viability was determined by addition of 10 BI of the tetrazolium salt MT]" (Sigma Chemical Co., St Louis, MO, USA) at 5 mg/ml in PBS. Plates were incubated at 37°C for 4 h, after which the supematant was removed and the dye reaction was stopped by addition of 200 gl of 0.04 N HC1 in isopropanol. After dissolution of the dye crystals, the resulting intensities were quantitated by absorbance at 570 nm. The titre of neutralization of each serum was defined as the reciprocal of the last dilution of the serum in the virus-serum mixture giving 50 % neutralization.
Statistical analysis The t test was used to compare the seroreactivity against V3 peptides, V3 IgG avidity and the neutralizing activity between the LS and AIDS children groups.
RESULTS Inhibition of syncytium formation by cell-tocell infection HIV 1 can spread by cell-to-cell transmission, and this pathway of infection leads to the formation of multinucleated giant cells or syncytia in vitro. Heat-inactivated sera were assessed for their capacity to inhibit syncytia formation between non-infected SupT1 target cells and H9 cells infected with different HIV 1 strains. Nine sera from the 11 LS children (82%) and 10 sera from the 16 AIDS children (62.5%) neutralized syncytia formation induced by HIV1My-infected H9 cells with titres up to 320 (table II). Moreover, neutralization titres were higher in LS children (mean 71, range 0-320) than in AIDS children (mean 41, range 0-160). In particular, a neutralization titre _> 40 was observed for 8 out of 11 sera from the LS children (73 %) and for only 5 out of 16 sera from the AIDS children (31%) (table I). By contrast, none of the sera inhibited syncytia formation induced by H9/HIV1LA I cells. As a control, uninfected Rwandan children were tested and showed no reactivity in this assay (titre < 20).
Neutralization of the cytopathic effect induced in MT4 cells Infection of MT4 cells with HIV1 resulted in a cytopathic effect leading to the formation of syncytia, followed by cell death. Heat-inactivated sera were then tested for their neutralization activity against the cell-free infection with HIV1MN, HIV1Rn and HIV1LAI by determination of cell viability. Table II shows that the highest neutralizing activity was observed when cells were infected with HIV1MN. Indeed, 100% of sera from LS children (11/11) and 81% of sera from AIDS children (13/16) gave neutralization titres up to 2560. We also observed that 8 (73%) and 10 (91%) sera from the LS group had neutralizing activity (titres b e t w e e n 40 and 640) against HIV1LA I and HIV1Rn, respectively. By comparison, serum samples of only 6 (37.5 %) and 13 (81%) of the AIDS group neutralized LAI and RII cell-free infection, respectively, with titres > 40. Moreover, the neutralizing activity against the three HIV 1 strains was higher in LS than in AIDS children, as determined by the number of children with titres > 320 against MN infection, and with titres > 80 against RII and LAI infection (table II). The means of the two groups were significantly different when cells were infected with HIV1MN (p = 0.05) and with HIVILAI (p < 0.01). Sera from the 4 HIVl-seronegative children had no neutralizing activity (titre < 20). The obviously greater neutralizing antibody titres for HIV1Rn compared t o HIVILA I is consistent with the African origin of the RII strain. However, the higher neutralization of HIV1MN infection suggests the prevalence of an MN-related strain in this part of Africa.
Seroreactivity against the PND peptides The seroreactivity was assessed against the synthetic peptide aa307-327 of the PND from different H I V I strains, and against US/European and African consensus V3 peptides. We observed that 100% of the sera f r o m the LS children (11/11) reacted against the V3AfCons2 peptide (fig. 1). A high percentage (91%) of sera from the LS children (10/11) were also reactive against
A N T I B O D I E S I N R W A N D A N C H I L D R E N W I T H L O N G - T E R M HIV1 I N F E C T I O N
T a b l e
205
II. Neutralizing activity of sera from LS and AIDS children. LS 11
Number of patients: Mean age in years (range)"
8.8
p value
AIDS 16 (6-11)
3.2
(1-5.5)
Mean neutralizing titres (range) Inhibition of syncytia formation induced by HIV 1MN-infected cells Number of children (%): with titre > 20 with titre _>40 Inhibition of the cytopathic effect induced in MT4 cells by HIV 1 MN Number of children (%): with titre > 40 with titre > 320 RII Number of children (%) : with titre > 40 with titre > 80 LAI Number of children (%) : with titre > 40 with titre > 80
(0-320)
71 9/11 8/11
869
41
(100) (73)
10/16 5/16
266
(40-2,560) 11/11 8/11
178 10/11 9/11 93 8/11 8/11
(0-160) (62.5) (31)
(0-2,560)
0.05
(81) (25) (0-320)
0.2
(81) (62.5) (0-160)
< 0.01
13/16 4/16
(100) (73) (0-640)
93 13/16 10/16
(91) (82) (20-160)
34 6/16 3/16
(73) (73)
0.3
(37.5) (19)
The neutralizing titre for each serum was the most representative value from at least 3 experiments. The mean titre for each group of children was calculated as the sum of the neutralizing titres divided by the number of children.
lOO
[
•
LS
I~1 AIDS
n=ll n=16
80
.~ 60
•~
40
20
0
-
AfCons2
MN
Cons
•
Z2
•
RF
LAI
V3MN and V 3 C o n s peptides. W e a k e r reactivity was observed for the sera from the A I D S children against the V 3 A f C o n s 2 (10/16, 62.5 %), V3MN (11/16, 69%) and V3Cons peptides (12/16, 75 %). B y c o n t r a s t , o n l y a f e w s e r a r e a c t e d against the V3m~ and V3z2 peptides, and no reactivity was o b s e r v e d t o w a r d the V3LA l p e p t i d e (fig. 1). A t a 1 0 0 - f o l d dilution, the r e a c t i v i t y against the three peptides (V3MN, V 3 C o n s and V 3 A f C o n s 2 ) was higher in serum samples f r o m L S children than f r o m A I D S children (data not s h o w n ) . T h e m e a n s o f O D for the t w o g r o u p s were significantly different in terms o f reactivity to the A f r i c a n c o n s e n s u s peptide (1.581 v e r s u s 0.995) (p < 0.01).
V3 peptides 1. Reactivity of sera from LS and AIDS children against the Noah-American/European (V3Cons) and the African (V3AfCons2) V3 consensus peptides, and V3 peptides from different HIV1 strains (MN, Z2, RF and LAI).
Fig.
All sera were tested at 1:100 dilution.
V3MN I g G
a v i d i t y
We have shown that sera from the LS children presented a h i g h e r neutralizing activity against
206
C. F R A I S I E R E T AL.
the HIV1MN strain. Sera were then tested for the avidity of the IgG directed against the V3M• peptide. Sera from 10 LS and 14 AIDS children were used at a dilution giving an OD of 1.0 +_0.1. The avidity index was higher in LS children (mean 70, range 35-95) than in AIDS children (mean 51, range 23-85) (fig. 2). In particular, 7 of the 9 sera from the LS children (78 %) and only 7 of the 14 sera from the AIDS children (50 %) had an avidity index > 50 (fig. 2). The means for the two groups were significantly different (p < 0.05).
DISCUSSION
Sixteen out of 537 children born to H I V I infected mothers and who were over 5 years old presented a prolonged course of HIV 1 infection despite a decrease in the CD4+/CD8 + ratio (Lepage et al., 1991). To find out the rationale for this p r o l o n g e d survival, we investigated the HIV 1-specific humoral response for 11 children, compared to 16 children who developed AIDS. The broadly neutralizing activity o f the LS children's sera, in particular against HIV1MN infection, was associated with reactivity against
the V3MN peptide, while low titre or no reactivity could be detected against the PND from the other strains. This is in agreement with other serological studies which showed the prevalence of V3MN-directed antibodies in m a n y parts of the world (Carrow et al., 1991 ; CheingsongP o p o v e t al., 1992; Warren et al., 1992). React i v i t y was also o b s e r v e d a g a i n s t b o t h the U S / E u r o p e a n and the African consensus V3 peptides, which present 83 and 70 % amino acid homologies with the V3MN peptide, respectively. Nevertheless, the LS children had stronger reactivity against these peptides and significantly higher V3MN IgG avidity than the AIDS children. Low V3IgG avidity could thus be associated with AIDS d e v e l o p m e n t , as has been shown for low p17 IgG avidity (Chargelegue et al., 1993). These data s h o w e d that the MNdirected humoral response can lead to neutralization o f different strains, in agreement with the r e s u l t s o f B e r m a n e t al. ( 1 9 9 2 ) , w h o observed that antibodies to LAI-rgpl20 neutralized only the homologous virus, whereas antibodies to MN-rgpl20 neutralized 6 out of 9 different isolates (Berman et al., 1992). Because syncytium formation is an important step that enables the virus to spread cell-to-cell (Brenner et al., 1991), the fact that the sera inhibited this
100 8O
8O
"~ 60
"~ 60
"= 40 "7.
:~ 40 "7,
20
20
2
3
4 5 6 7 LS children
8
9
10
1 2 3 4 5 6 7 8 9 1011 12 13 14 AIDS children
Fig. 2. Avidity index of the IgG response to HIVluN V3 peptide in sera from LS and AIDS children. The means for the two groups were significantly different (p < 0.05).
A N T I B O D I E S I N R W A N D A N C H I L D R E N W I T H L O N G - T E R M HIV1 I N F E C T I O N
process suggests that the presence of syncytiainhibiting antibodies might be responsible for the slow course of HIV 1 infection in these children. Since the V3 loop was identified as a fusion domain (Freed et al., 1991), essential for gpl20-mediated syncytia formation (Travis et al., 1992), the presence in surviving infected children of high levels of MN-like/PND-specific antibodies supports the notion that the humoral response to the V3 region of HIV1 gpl20 may contribute to protection against disease progression. Indeed, previous studies have described the prevalence of V3-directed antibodies in long-term asymptomatic carriers compared with that of AIDS patients, and have provided evidence for declining levels of these antibodies during the course of infection (Neurath et al., 1990). Moreover, it has recently been reported that the majority of neutralizing antibodies in H I V l - i n f e c t e d p a t i e n t s r e c o g n i z e V3 loop sequences (Vogel et al., 1994). The gpl20 PND lies within a loop formed by a disulphide bridge between two conserved cysteines which play a role in maintaining its tertiary structure. Antibodies directed against this peptide might then alter the protein conformation essential for biological activity (Rossi et al., 1989). Moreover, recent studies indicated that anti-V3 m A b s could neutralize several HIV1 isolates. These broadly neutralizing antibodies were obtained by i m m u n i z a t i o n with HIV1MN V3 peptides (Ohno et al., 1991 ; White-Scharf et al., 1993) and d e f i n e d by their reactivity against the HIV1MN V3 loop (Gorny et aL, 1992). However, none of our sera cross-neutralizing MN and LAI were reactive in ELISA with the V3LAI peptide. As observed by others ( N k e n g a s o n g et al., 1994), our data suggest that there are conserved neutralizing epitopes outside the V3 loop. These epitopes could, in particular, be part of the CD4binding domain of gpl20, whose specific antibodies could neutralize divergent HIV 1 isolates (Steimer et al., 1991). Moreover, a loss of antibodies directed against the CD4-binding site has been associated with rapid disease progression (Cavacini et al., 1993). A previous study of these perinatally HIV 1infected children revealed the presence of p17 IgG and persistent production of IgM to HIV 1
207
envelope glycoproteins, as compared to 16 children with paediatric AIDS (Van de Perre et al., 1992). In the present study, we showed that, despite their small number, sera from these LS children, compared to sera from the AIDS children, had higher neutralizing antibody titres against different HIV1 strains. They also had higher levels of antibodies directed against MNlike V3 peptides, as well as higher V3MN IgG avidity, which could contribute to the prolonged survival of these LS children. We have also observed the presence of non-syncytium-inducing isolates that could be neutralized by the autologous serum, for 4 of the LS children. Delayed HIV1 isolation in culture was also obtained for 2 of them (data not shown). Cell samples were not available for other LS or AIDS children, but these data are in agreement with previous studies showing a correlation between the biological phenotype of HIV1 isolates and the clinical course of infection (De Rossi et al., 1991; Koot et al., 1992; Tersmette et al., 1989). Although sera should be tested for their neutralizing activity against syncytium formarion, as well as against the cytopathic effect induced by a field isolate from Rwanda, our results concerning the humoral response suggest the prevalence of an antigenically MN-related strain in Rwanda, as described in other countries of Africa (Albert et al., 1992; Carrow et al., 1991; Warren et al., 1992). Moreover, because of the strong seroreactivity observed against the A f r i c a n c o n s e n s u s V3 p e p t i d e , we c a n n o t exclude the importance of an African HIV 1 isolate in vaccine development. Nevertheless, the homology of the HIV1MN V3 region with both US/European and African consensus sequences should also be considered in vaccination strategies in this part of Africa.
Acknowledgments This work was supported by the Institut National de la Sant6 et de la Recherce Mrdicale (INSERM) and the Agence Nationale de Recherche sur le SIDA (ANRS), France. C. Fraisier was a recipient from Fondation Marcel Merieux. We wish to thank Drs. V. Boyer and B. Verrier for advice and criticism.
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C. F R A I S I E R E T AL.
Anticorps dirig6s contre la r6gion V3 proche de la souche MN et neutralisant diff6rentes souches de VIH1, chez des enfants rwandais infect~s par le VIH1 et pr6sentant une survie prolong6e Les s6rums de 11 enfants rwandais infect6s de faqon p6rinatale par le VIH1 et pr6sentant une survie prolong6e (<~ ou LS) ont 6t6 test6s in vitro pour la pr6sence d'anticorps neutralisant d i f f 6 r e n t e s s o u c h e s de V I H 1 . Ces 11 s 6 r u m s d'enfants LS ont 6t6 compar6s aux s6rums de 16 enfants rwandais atteints de SIDA. Les s6rums des enfants infect6s par le VIH1 ont rnontr6 la plus forte activit6 neutralisante contre l'infection par le virus libre de la s o u c h e MN. Ils inhibent 6 g a l e m e n t l'infection par le virus fibre des souches RII et LAI des titres plus faibles. Des titres de neutralisation plus forts ont 6t6 observ6s pour les s6rums des enfants LS compar6s aux titres des s6rums des enfants atteints de SIDA, avec une diff6rence significative pour les titres contre les souches M N et LAI. Les s6rums des enfants LS inhibent 6galement la formation des syncytia induits par les cellules H9 infect6es par MN, avec des titres de neutralisation plus 61ev6s que ceux des s6rums des enfants atteints de SIDA. Les s6rums des enfants infect6s par le VIH1 ont rnontr6 une r6activit6 contre le peptide V3MN, ainsi que contre les peptides V3 consensus issus de s6quences nordam6ricaines et africaines. Une plus forte r6activit6 a 6t6 observ6e pour les s6rums des enfants LS par rapport ~ celle des s6rums des enfants atteints de SIDA, avec une diff6rence significative contre le peptide V3 c o n s e n s u s africain. Les e n f a n t s LS p r 6 s e n t e n t 6galement une avidit6 des IgG anti-V3MN significativement plus 61ev6e que celle des enfants atteints de SIDA. Ces donn6es supportent la notion que la r6ponse h u m o r a l e dirig6e contre la r6gion V3, associ6e ?~une activit6 neutralisante contre diff6rentes souches de VIH1, pourrait &re un facteur important pour la survie prolong6e de ces enfants. La r6ponse dirig6e contre la souche MN sugg~re par ailleurs qu'une souche antig6niquement proche de MN pourrait ~tre pr6valente au Rwanda et que des s6quences V3 proches de MN pourraient ~tre incluses dans les candidats-vaccins dans cette partie d'Afrique. Mots-cl~s: HIV1, SIDA, Enfant, IgG; Rwanda, Infection p6rinatale, Survie prolong6e, HIV1, N' Boucle V3, Anticorps neutralisants, Avidit6 ~es IgG, Candidats-vaccins.
References Albert, J., Franzen, L., Jansson, M., Scarlatti, G. Kataaha, P.K., Katabira, E., Mubiro, F., Rydaker, M., Rossi,
P., Pettersson, U. & Wigzell, H. (1992), Ugandan HIV-1 V3 loop sequences closely related to the US/European consensus. Virology, 190, 674-681. Bergeron, L., Sullivan, N. & Sodroski, J. (1992), Target cell-specific determinants of membrane fusion within the human immunodeficiency virus type 1 gpl20 third variable region and gp41 amino terminus. J. Virol., 66, 2389-2397. Berman, P.W., Matthews, T.J., Rddle, L., Champe, M., Hobbs, M.R., Nakamura, G.R., Mercier, J., Eastman, D.J., Lucas, C., Langlois, A.J., Wurm, F.M. & Gregory, T.J. (1992), Neutralization of multiple laboratory and clinical isolates of human immunodeficiency virus type 1 (H1V-1) by antisera raised against gpl20 from the MN isolate of HIV-1. J. Virol., 66, 4464-4469. Blanche, S., Tardieu, M., Duliege, A.M. et al. (1990), Longitudinal study of 94 symptomatic infants with perinatally acquired human immunodeficiency virus infection. Evidence for a bimodal expression of clinical and biological symptoms. A.J.D.C., 144, 12101214. Bowen, D.L., Lane, H.C. & Fauci, A.S. (1985), Immunologic abnormalities in the acquired immunodeficiency syndrome. Annu. Rev. Immunol., 3,477-500. Brenner, T.J., Dahl, K.E., Olson, B., Miller, G. & Andiman, W.A. (1991), Relation between I-~V-1 syncytium inhibition antibodies and clinical outcome in children. Lancet, 337, 1001-1005, Broliden, P.A., Gegerfelt, A., Clapham, P., Rosen, J. Fenyo, E.M., Wahren, B. & Broliden, K. (1992), Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins. Proc. Natl. Acad. Sci. USA., 89, 461-465. Carrow, E.W., Vujcic, L.K., Glass, W.L., Seamon, K.B., Rastogi, S.C., Hendry, R.M., Boulos, R., Nzila, N. & Quinnan Jr, G.V. (1991), High prevalence of antibodies to the V3 region principal neutralizing determinant of HIV-IMN in sera from Africa and the Americas. AIDS. Res. Hum. Retroviruses., 7, 831-838. Cavacini, L.A., Emes, C.L., Power, J., Underdahl, J., Goldstein, R., Mayer, K. & Posner, M.R. (1993), Loss of serum antibodies to a conformational epitope of HIV-1/gpl20 identified by a monoclonal antibody is associated with disease progression. J. Acquired lmune Defic. Syndr., 6, 1093-1102. Chargelegue, D., Colvin, B.T. & O'Toole, C.M. (1993), A 7-year analysis of anti-Gag (p17 and p24) antibodies in HIV-l-seropositive patients with haemophilia: immunoglobulin G titre and avidity are earlier predictors of clinical course. AIDS, 7, $87-$90. Cheingsong-Popov, R., Callow, D., Beddows, S., Shaunak, S., Wasi, C., Kaleebu, P., Giiks, C., Petrascu, I.V., Garaev, M., Watts, D.M., Constantine, N.T. & Weber, J.N. (1992), Geographic diversity of human immunodeficiency virus type 1: serologic reactivity to env epitopes and relationship to neutralization. J. Infect. Dis., 165, 256-261. Chesebro, B., Wehrly, K., Nishio, J. & Perryman, S. (1992), Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unsuai V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism. J. ViroL, 66, 6547-6554.
A N T I B O D I E S I N R W A N D A N C H I L D R E N W I T H L O N G - T E R M HIV1 I N F E C T I O N
De Jong, J.J., Goudsmit, J., Keulen, W., Klaver, B., Krone, W., Tersmette, M. & De Ronde, A. (1992), Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity. J. Virol., 66, 757-765. De Rossi, A., Pasti, M., Mammano, F., Ometto, L., Giaquinto, C. & Chieco-Bianchi, L. (1991), Perinatal infection by human immunodeficiency virus type 1 (HIV-1): relationship between proviral copy number in vivo, viral properties in vitro, and clinical outcome. J. Med. Virol., 35, 283-289. Emini, E.A., Schleif, W.A., Nunberg, J.H., Conley, A.J., Eda, Y., Tokiyoshi, S., Putney, S.D., Matsushita, S., Cobb, K.E., Jette, C.M., Eichberg, J.W. & Murthy, K.K. (1992), Prevention of HIV-1 infection in chimpanzees by gpl20 V3 domain-specific monoclonal antibody. Nature (Lond.), 355, 728-730. Fallon, J., Eddy, J., Wiener, L. & Pizzo, P.A. (1989), Human immunodeficiency virus in children. J. Pediatr., 114, 1-30. Fouchier, R.A., Groenink, M., Kootstra, N.A., Tersmette, M., Huisman, H.G., Miedema, F. & Schuitemaker, H. (1992), Phenotype-associated sequence variation in the third variable domain of human imunodeficiency virus type 1 gpl20 molecule. J. Virol., 66, 3183-3187. Freed, E.O., Myers, D.J. & Risser, R. (1991), Identification of the principal neutralizing determinant of human immunodeficiency virus type 1 as a fusion domain. J. Virol., 65, 190-194. Gomy, M.K., Conley, A.J., Karwowska, S., Buchbinder, A., Xu, J.Y., Emini, E.A., Koenig, S. & Zolla-Pazner, S. (1992), Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. J. Virol., 66, 7538-7542. Hwang, S.S., Boyle, T.J., Lyerly, H.K. & Cullen, B.R. (1991), Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science, 253, 71-74. Javaherian, K., Langlois, A.J., McDanal, C., Ross, K.L., Eclder, L.I., Jellis, C.L., Profy, A.T., Rusche, J.R., Bolognesi, D.P., Putney, S.D. & Matthews, T.J. (1989), Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Natl. Acad. Sci. USA, 86, 6768-6772. Koot, M., Vos, A.H.V., Keet, R.P.M., De Goede, R.E.Y., Dercksen, M.W., Terpstra, F.G., Coutinho, R.A., Miedema, F. & Tersmette, M. (1992), HIV-1 biological phenotype in long-term infected individuals evaluated with an MT2 cocultivation assay. AIDS, 6, 4954. LaRosa, G.J., Davide, J.P., Weinhold, K., Waterbury, J.A., Profy, A.T., Lewis, J.A., Langlois, A.J., Dreesman, G.R., Boswell, R.N., Shadduck, P., Holley, L.H., Karplus, M., Bolognesi, D.P., Matthews, T.J., Emini, E.A. & Putney, S.D. (1990), Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science, 249, 932-935. Lepage, P., Van de Perre, P., van Vliet, G., Nsengumuremyi, F., Van Goethem, C., Kestelyn, P., Msellati, P. & Hitinama, D.G. (1991), Clinical and endocrinologic manifestation in perinatally human immunodeficiency virus type-l-infected children aged 5 years or older. A.J.D.C., 145, 1248-1251. Levy, J.A. (1993), HIV pathogenesis and long-term survival. AIDS, 7, 1401-1410.
209
Myers, G., Berzofsky, J.A., Rabson, A.B., Smith, T.F. & Wong-Staal, F. (1991), Human retroviruses and AIDS, in "Theoretical biology and biophysics". Los Alamos National Laboratory, Los Alamos. Neurath, A.R., Strick, N., Taylor, P., Rubinstein, P. & Stevens, C.E. (1990), Search for epitope-specific antibody responses to human immunodeficiency virus (HIV-1) envelope glycoproteins signifying resistance to disease development. AIDS Res. Hum. Retroviruses., 6, 1183-1193. Nkengasong, J.N., Peeters, M., Ndumbe, P., Janssens, W., Willems, B., Fransen, K., Ngolle, M., Piot, P. & van der Groen, G. (1994), Cross-neutralizing antibodies to HIV-1ArzrT0and H1V-lnm in sera of African and Belgian HIV-l-infected individuals. AIDS, 8, 1089-1096. Ohno, T., Terada, M., Yoneda, Y., Shea, K.W., Chambers, R.F., Stroka, D.M., Nakamura, M. & Kufe, D.W. (1991), A broadly neutralizing monoclonal antibody that recognizes the V3 region of human immunodeficiency virus type 1 glycoprotein gpl20. Proc. Natl. Acad. Sci. USA., 88, 10726-10729. Oxtoby, M.J. (1990), Perinatally acquired HIV infection. Pediatr. Infect. Dis. J., 9, 609-619. Robertson, G.A., Kostek, B.M., Schleif, W.A., Lewis, J.A. & Emini, E.A. (1988), A microtiter cell-culture assay for the determination of anti-human immunodeficiency virus neutralizing antibody activity. J. Virol. Methods., 20, 195-202. Rossi, P., Moschese, V,, Broliden, P.A., Fundaro, C., Quinti, I., Plebani, A., Giaquinto, C., Tovo, P.A., Ljunggren, K., Rosen, J., WigzeU, H., Jondal, M. & Wahren, B. (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. Safrit, J.T., Fung, M.S.C., Andrews, C.A., Braun, D.G., Sun, W.N.C., Chang, T.W. & Koup, R.A. (1993), huPBL-SC1D mice can be protected from HIV-I infection by passive transfer of monoclonal antibody to the principal neutralizing determinant of envelope gpl20. AIDS, 7, 15-21. Steimer, K.S., Scandella, C.J., Skiles, P.V. & Haigwood, N.L. (1991), Neutralization of divergent HIV-I isolates by conformation-dependent human antibodies to gpl20. Science., 254, 105-108. Tersmette, M., Gruters, R.A., De Wolf, F., De Goede, R.E.Y., Lange, J.M.A., Schellekens, P.T.A., Goudsmit, J., Huisman, H.G. & Miedema, F. (1989), Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential isolates. J. Virol., 63, 2118-2125. Tovo, P.A., De Martino, M., Gabiano, C., Cappello, N., D'Elia, R., Loy, A., Plebani, A., Zuccotti, G.V., Dallacasa, P., Ferraris, G., Caselli, D., Fundaro', C., D'Argenio, P., Galli, L., Princip, N., Stegagno, M., Ruga, E., Palomba, E. et al. (1992), Prognostic factors and survival in children with perinatal HIV-I infection. Lancet, 339, 1249-1253. Travis, B.M., Dykcrs, T.I., Hewgill, D., Ledbetter, J., Tsu, T.T., Hu, S.L. & Lewis, J.B. (1992), Functional role of the V3 hypervariable region of HIV-1 gpl60 in the processing of gp160 and in the formation of syncytia in CD4+ cells. Virology, 186, 313-317.
210
C. F R A I S I E R E T AL.
Van de Perre, P., Lepage, P., Simonon, A., Desgranges, C., Hitimana, D.G., Bazubagira, A., Van Goethem, C., Kleinschmidt, A., Bex, F., Brolinden, K., Erfle, V., Gotch, F. & Bumy, A. (1992), Biological markers associated with prolonged survival in African children maternally infected by the human immunodeficiency virus type 1. AIDS. Res. Hum. Retroviruses., 8, 435-442. Vogel, T., Kurth; R. & Norley, S. (1994), The majority of neutralizing abs in HIV-l-infected patients recognize linear V3 loop sequences. Studies using HIV-I(MN) multiple antigenic peptides. J. Immunol., 153, 18951904. Warren, R.Q., Nkya, W.M., Shao, J.F., Anderson, S.A., Wolf, H., Hendrix, C.W., Kanda, P., Wabuke,
M., Boswell, R.N., Redfield, R.R. & Kennedy, R.C. (1992), Comparison of antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gpl60 epitopes in sera from HIV-l-infected individuals from Tanzania and from the United States. J. Clin. Microbiol., 30, 126-131. White-Scharf, M.E., Potts, B.J., Smith, M., Sokolowski, K.A., Rusche, J.R. & Silver, S. (1993), Broadly neutralizing monoclonal antibodies to the V3 region of HIV-1 can be elicited by peptide immunization. Virology, 192, 197-206. World Health Organization. (1986), Acquired immunodeficiency syndrom (AIDS). Weekly Epidemiological Record., 61, 69-73.
Res. Virol. 1995, 146, 201-210
Broadly neutralizing, MN-like PND-directed antibodies in Rwandan children with long-term HIV1 infection C. Fraisier (~) (*), P. Van de Perre (2), p. Lepage (3), D.G. Hitimana (3), E. Karita (2) and C. Desgranges (~) (**~ (I) INSERM U271, 151 Cours A. Thomas, 69424 Lyon Cedex 03 (France), (2) National A1DS Control Programme Ministry of Health, Kigali (Rwanda), and (~) Centre Hospitalier de Kigali, Kigali (Rwanda)
SUMMARY
Sera from 11 perinatally HIVl-infected Rwandan children with prolonged survival were tested in vitro for the presence of neutralizing antibodies against different HIV1 strains. These 11 sera from long survivor (LS) children were compared with 16 sera from Rwandan children with AIDS. Sera from HIVl-infected children exhibited the greatest neutralizing activity against HIVIMN cell-free infection. They also inhibited HIVIRu and HIVIL~ cell-free infection with lower titres. Higher neutralization titres were observed in sera-from LS compared to the AIDS group, with a significant difference for HIVIMN and HIVIL~J strains. Sera from LS children also inhibited syncytium formation induced by HIVIMN-infected cells with higher titres than AIDS children. Sera from the HIVl-infected children showed reactivity to the HIVIMN V3 peptide, as well as to both the US/European and the African consensus V3 peptides. Higher reactivity was observed in sera from LS than from AIDS children, and the difference was significant toward the African consensus peptide. The LS children also had significantly higher V3MN IgG avidity than the AIDS children. These data support the notion that the humoral response to the V3 domain, associated with a broadly neutralizing activity, may be an important factor in the prolonged survival of these children. The specificity against HIVIMN also suggests that an antigenically MN-related strain may be prevalent in Rwanda, and that an MN-related principal neutralizing domain sequence could be an important determinant for candidate vaccines in this part of Africa. Key-words: HIV1, AIDS, Child, IgG; Rwanda, Perinatal infection, Long-term survival, HlVlMN, V3 loop, Neutralizing antibodies, IgG avidity, Vaccination strategies.
INTRODUCTION Infection with human immunodeficiency virus type 1 (HIV1) leads to profound, progressive impairment of T-cell function and to the develop-
ment of the acquired i m m u n o d e f i c i e n c y syndrome (AIDS) (Bowen et al., 1985). HIV1 infection has been shown to progress more rapidly in children than in adults (Fallon et al., 1989; Oxtoby, 1990). H o w e v e r , p e r i n a t a l l y HIV1-
Submitted November 11, 1994, accepted January 25, 1995. (*) Presentaddress: Lab. of Gene Structureand Expression,NationalInstitutefor Medical Research,The Ridgeway,Mill Hill, London NW71AA,UK. (**) Correspondingauthor.
C. FRAISIER E T AL.
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infected children surviving to over 5 years of age have recently been reported (Blanche et al., 1990; Lepage et al., 1991; Tovo et al., 1992). We described the prolonged course of HIV 1 infection in 16 African children maternally infected by HIV1 (Lepage et al., 1991). Most of the children who survived beyond 5 years presented with moderate signs and symptoms. Numerous biological markers have been associated with long-term survival, such as low viral load, the presence of non-syncytium-inducing and monocytotropic HIV1 strains and HIVl-specific CD8 + T-cell immune responses (Levy, 1993). In addition to the cellular response, neutralizing antibodies directed against HIV1 envelope glycoproteins could be an important component of the protective immune response. We previously reported that prolonged survival was associated with persistent production of IgM to HIV 1 envelope proteins and of IgG to p17 (Van de P e r r e e t al., 1992). Antibodies from HIVl-infected individuals could neutralize a broad variety of HIV1 isolates. These neutralizing antibodies, directed against conformational epitopes of g p l 2 0 , block the gpl20 interaction with the CD4 receptor (Steimer et al., 1991), and a decline in antibodies to the CD4-binding site has been correlated with rapid disease progression (Cavacini et al., 1993). Furthermore, four major neutralizing regions of HIV1 glycoproteins have been identified (Broliden et al., 1992), and a n t i b o d i e s against restricted epitopes of gpl20 and gp41 have been shown to be significantly less prevalent in AIDS than in asymptomatic patients (Neurath et al., 1990). In particular, a correlation has been demonstrated between the level of antibodies to the aa303-338 region and development of the disease (Neurath et al., 1990). This region, located in the third hypervariable region of gpl20 (V3), forms a loop between two conserved cysteines at positions 303 and 338, and has been determined to be the principal neutralizing domain (PND) of HIV1
LS mAb NBCS PBS-T
= = = =
l o n g survivor. monoclonal antibody. new-born calf serum. p h o p h a t e - b u f f e r e d saline + T w e e n - 2 0 .
(Javaherian et al., 1989). The PND has also been identified as a fusion domain (Freed et al., 1991) and is essential for gp 120-mediated syncytia formation (Travis et al., 1992). It also contains target-cell-specific determinants of membrane fusion (Bergeron et al., 1992) and tropism for T cells and macrophages (Chesebro et al., 1992; Hwang et al., 1991). The V3 loop was also related to both syncytium-inducing and replicating phenotypes of HIV1 (De Jong et al., 1992; F o u c h i e r et al., 1992). Antibodies directed against linear epitopes of the V3 loop are generally defined as type-specific and react with individual viral isolates. However, the neutralization of diverse HIV1 isolates could be observed with monoclonal antibodies (mAb) directed against the V3 domain. These broadly neutralizing antibodies were, in particular, obtained by immunization with HIVIMN V3 peptides (Ohno et al., 1991 ; White-Scharf et al., 1993) or defined by their reactivity against the HIV1MN V3 loop (Gorny et al., 1992). These anti-V3 antibodies are able to block infectivity in vitro, and it has also been shown that g p l 2 0 V 3 - d o m a i n - s p e c i f i c mAbs could prevent HIV 1 infection in chimpanzees (Emini et al., 1992) as well as in hu-PBLSCID mice (Safrit et aL, 1993). The results presented here reveal the presence of broadly neutralizing activity and MN-like PND-specific antibodies with higher V3MN IgG avidity in sera from long-term survivor (LS) children. This activity could be involved in the less severe course of HIV1 disease in these children.
PATIENTS
AND
METHODS
Patients
A group of 16 out of 537 children born to HIV1infected mothers and more than 5 years old at enrollment were diagnosed at the Paediatric Department of the Centre Hospitalier de Kigali, Kigali, Rwanda.
PND RT Sn
= = =
principal neutralizing domain. room temperature. seronegative.
ANTIBODIES IN RWANDAN
CHILDREN WITH LONG-TERM HIV1 INFECTION
Sera from 11 out of these 16 children (LS) were collected in February and March 1990. These sera were compared in our study with sera from 16 Rwandan children 18 months to 6 years old who fulfilled the World Health Organization definition of paediatric AIDS (WHO, 1986) (AIDS) and 4 sera from H I V I seronegative (Sn) Rwandan children 6 to 7 years old.
Virus stock Virus suspensions of HIV1 strains were obtained f r o m supernatants of HIV1MN ( N o r t h - A m e r i c a ) , HIV1LM (Europe) and HIV1Ru (Zaire) chronically infected H9 cells provided by the National Institutes of Health (NIH), passed through 0.45-Bm filters and stored at - 8 0 ° C before use. For each virus stock, a MT4-cell infectivity endpoint was defined as the last dilution of the virus stock that killed 100% MT4 cells in a 7-day assay (Gorny et al., 1992; Robertson et al., 1988).
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tide at 1 p.g/ml in 0.1 M carbonate buffer pH 9.5. Plates w e r e w a s h e d t w i c e with PBS c o n t a i n i n g 0.1% Tween-20 (PBS-T), saturated for 30 min at 37°C with 200 ~1 o f PBS + 10% n e w - b o r n c a l f serum (NBCS) and incubated for 1 h at room temperature (RT) with 100 ~tl of sera at 1:100 dilution in 5 x P B S - T - 1 0 % NBCS. After extensive washing, bound antibodies were detected with peroxidaseconjugated goat anti-human immunoglobulins for 1 h at RT. Then, the enzymatic reaction was developed with the substrate, O-phenylenediamine, and read at 492 nm with a "Titertek Multiscan" (Flow Laboratories, Irvine, Scotland, UK). Cut-off values were set at 3 SD plus mean absorbance of the four negative controls. For the V3MN IgG avidity measurement, 100 gtl of the serum dilution giving an OD of 1.0 +_ 0.1 was d i s s o c i a t e d f r o m the V3MN solid p h a s e at 100 ng/well with 100 I11 of 8 M urea. The avidity index was calculated as (OD with 8 M urea/OD without urea) × 100.
Cell-to-cell syncytium formation inhibition assay Synthetic peptides Peptides corresponding to the g p l 2 0 PND were kindly p r o v i d e d by the A g e n c e N a t i o n a l e de la Recherche sur le S I D A and were synthesized by Neosystem Laboratories (Strasbourg, France). Consensus V3 peptides were kindly provided by Dr F. Barin and Dr A. Baillou (URA CNRS 1334, Tours, France). The peptide referred to as V3Cons (table I) c o r r e s p o n d e d to the U S / E u r o p e a n c o n s e n s u s sequence determined by Larosa et al. (1990). The peptide referred to as V3AfCons2 corresponded to the African consensus sequence derived from the V3 sequences of 108 African isolates (Myers et al., 1991).
Serial 2-fold dilutions (starting at 1:20) of heatinactivated sera (100 ~tl) were incubated in a microplate with 50 ~tl of HIV1LM- or HIV1MN-infected H9 cells at 10~cells/ml for 1 h at 37°C, 5 % CO 2. Fifty microliters of non-infected SupT1 target cells at 2 × 105 cells/ml were then added. After 24 h of culture at 37°C, syncytia were enumerated by light microscopy. In the control wells without serum, 50 to 80 multinucleated giant cells were observed. The neutralization titre was defined as the reciprocal of the highest dilution resulting in 5 0 % reduction in the number of syncytia when compared to the control wells without serum.
Neutralization of the cytopathic effect ELISA Microtitre plates (Immulon 2, Dynatech Laboratories Inc, Chantilly, VA, USA) were coated overnight at 37°C with 100 B1 of the V3 synthetic pep-
This assay was performed as described elsewhere ( R o b e r t s o n et al., 1988). Serial 2-fold dilutions (starting at 1:20) of heat-inactivated sera (50 pJ) were incubated for 1 h at r o o m temperature with
Table I. V3 loop sequences. MN V3Cons LAI RF Z2 V3AfCons2
CNKRKRIHI G NTRKSIHI G CNTRKS I R I QRG CNTRKS I TKG CNIRQRTPI G NTRQRTRI G
PGRAFYTT PGRAFYPGR AFVPGRVI YLGQSLYPGQAFY-
KN T TGE I IGDI T IGK A TGQ T TKTRS T TGI I GD
RQAHC
I RQAHC
204
C. FRAISIER E T AL.
50 pl of HIV1MN, HIV1LAI or HIV1Rn viral supernatants at the last dilution giving 100% cell killing (endpoint dilution). MT4 cells (5 × 10 4 cells in 100 ~tl) were added. After a 7-day incubation at 37°C, cell viability was determined by addition of 10 BI of the tetrazolium salt MT]" (Sigma Chemical Co., St Louis, MO, USA) at 5 mg/ml in PBS. Plates were incubated at 37°C for 4 h, after which the supematant was removed and the dye reaction was stopped by addition of 200 gl of 0.04 N HC1 in isopropanol. After dissolution of the dye crystals, the resulting intensities were quantitated by absorbance at 570 nm. The titre of neutralization of each serum was defined as the reciprocal of the last dilution of the serum in the virus-serum mixture giving 50 % neutralization.
Statistical analysis The t test was used to compare the seroreactivity against V3 peptides, V3 IgG avidity and the neutralizing activity between the LS and AIDS children groups.
RESULTS Inhibition of syncytium formation by cell-tocell infection HIV 1 can spread by cell-to-cell transmission, and this pathway of infection leads to the formation of multinucleated giant cells or syncytia in vitro. Heat-inactivated sera were assessed for their capacity to inhibit syncytia formation between non-infected SupT1 target cells and H9 cells infected with different HIV 1 strains. Nine sera from the 11 LS children (82%) and 10 sera from the 16 AIDS children (62.5%) neutralized syncytia formation induced by HIV1My-infected H9 cells with titres up to 320 (table II). Moreover, neutralization titres were higher in LS children (mean 71, range 0-320) than in AIDS children (mean 41, range 0-160). In particular, a neutralization titre _> 40 was observed for 8 out of 11 sera from the LS children (73 %) and for only 5 out of 16 sera from the AIDS children (31%) (table I). By contrast, none of the sera inhibited syncytia formation induced by H9/HIV1LA I cells. As a control, uninfected Rwandan children were tested and showed no reactivity in this assay (titre < 20).
Neutralization of the cytopathic effect induced in MT4 cells Infection of MT4 cells with HIV1 resulted in a cytopathic effect leading to the formation of syncytia, followed by cell death. Heat-inactivated sera were then tested for their neutralization activity against the cell-free infection with HIV1MN, HIV1Rn and HIV1LAI by determination of cell viability. Table II shows that the highest neutralizing activity was observed when cells were infected with HIV1MN. Indeed, 100% of sera from LS children (11/11) and 81% of sera from AIDS children (13/16) gave neutralization titres up to 2560. We also observed that 8 (73%) and 10 (91%) sera from the LS group had neutralizing activity (titres b e t w e e n 40 and 640) against HIV1LA I and HIV1Rn, respectively. By comparison, serum samples of only 6 (37.5 %) and 13 (81%) of the AIDS group neutralized LAI and RII cell-free infection, respectively, with titres > 40. Moreover, the neutralizing activity against the three HIV 1 strains was higher in LS than in AIDS children, as determined by the number of children with titres > 320 against MN infection, and with titres > 80 against RII and LAI infection (table II). The means of the two groups were significantly different when cells were infected with HIV1MN (p = 0.05) and with HIVILAI (p < 0.01). Sera from the 4 HIVl-seronegative children had no neutralizing activity (titre < 20). The obviously greater neutralizing antibody titres for HIV1Rn compared t o HIVILA I is consistent with the African origin of the RII strain. However, the higher neutralization of HIV1MN infection suggests the prevalence of an MN-related strain in this part of Africa.
Seroreactivity against the PND peptides The seroreactivity was assessed against the synthetic peptide aa307-327 of the PND from different H I V I strains, and against US/European and African consensus V3 peptides. We observed that 100% of the sera f r o m the LS children (11/11) reacted against the V3AfCons2 peptide (fig. 1). A high percentage (91%) of sera from the LS children (10/11) were also reactive against
A N T I B O D I E S I N R W A N D A N C H I L D R E N W I T H L O N G - T E R M HIV1 I N F E C T I O N
T a b l e
205
II. Neutralizing activity of sera from LS and AIDS children. LS 11
Number of patients: Mean age in years (range)"
8.8
p value
AIDS 16 (6-11)
3.2
(1-5.5)
Mean neutralizing titres (range) Inhibition of syncytia formation induced by HIV 1MN-infected cells Number of children (%): with titre > 20 with titre _>40 Inhibition of the cytopathic effect induced in MT4 cells by HIV 1 MN Number of children (%): with titre > 40 with titre > 320 RII Number of children (%) : with titre > 40 with titre > 80 LAI Number of children (%) : with titre > 40 with titre > 80
(0-320)
71 9/11 8/11
869
41
(100) (73)
10/16 5/16
266
(40-2,560) 11/11 8/11
178 10/11 9/11 93 8/11 8/11
(0-160) (62.5) (31)
(0-2,560)
0.05
(81) (25) (0-320)
0.2
(81) (62.5) (0-160)
< 0.01
13/16 4/16
(100) (73) (0-640)
93 13/16 10/16
(91) (82) (20-160)
34 6/16 3/16
(73) (73)
0.3
(37.5) (19)
The neutralizing titre for each serum was the most representative value from at least 3 experiments. The mean titre for each group of children was calculated as the sum of the neutralizing titres divided by the number of children.
lOO
[
•
LS
I~1 AIDS
n=ll n=16
80
.~ 60
•~
40
20
0
-
AfCons2
MN
Cons
•
Z2
•
RF
LAI
V3MN and V 3 C o n s peptides. W e a k e r reactivity was observed for the sera from the A I D S children against the V 3 A f C o n s 2 (10/16, 62.5 %), V3MN (11/16, 69%) and V3Cons peptides (12/16, 75 %). B y c o n t r a s t , o n l y a f e w s e r a r e a c t e d against the V3m~ and V3z2 peptides, and no reactivity was o b s e r v e d t o w a r d the V3LA l p e p t i d e (fig. 1). A t a 1 0 0 - f o l d dilution, the r e a c t i v i t y against the three peptides (V3MN, V 3 C o n s and V 3 A f C o n s 2 ) was higher in serum samples f r o m L S children than f r o m A I D S children (data not s h o w n ) . T h e m e a n s o f O D for the t w o g r o u p s were significantly different in terms o f reactivity to the A f r i c a n c o n s e n s u s peptide (1.581 v e r s u s 0.995) (p < 0.01).
V3 peptides 1. Reactivity of sera from LS and AIDS children against the Noah-American/European (V3Cons) and the African (V3AfCons2) V3 consensus peptides, and V3 peptides from different HIV1 strains (MN, Z2, RF and LAI).
Fig.
All sera were tested at 1:100 dilution.
V3MN I g G
a v i d i t y
We have shown that sera from the LS children presented a h i g h e r neutralizing activity against
206
C. F R A I S I E R E T AL.
the HIV1MN strain. Sera were then tested for the avidity of the IgG directed against the V3M• peptide. Sera from 10 LS and 14 AIDS children were used at a dilution giving an OD of 1.0 +_0.1. The avidity index was higher in LS children (mean 70, range 35-95) than in AIDS children (mean 51, range 23-85) (fig. 2). In particular, 7 of the 9 sera from the LS children (78 %) and only 7 of the 14 sera from the AIDS children (50 %) had an avidity index > 50 (fig. 2). The means for the two groups were significantly different (p < 0.05).
DISCUSSION
Sixteen out of 537 children born to H I V I infected mothers and who were over 5 years old presented a prolonged course of HIV 1 infection despite a decrease in the CD4+/CD8 + ratio (Lepage et al., 1991). To find out the rationale for this p r o l o n g e d survival, we investigated the HIV 1-specific humoral response for 11 children, compared to 16 children who developed AIDS. The broadly neutralizing activity o f the LS children's sera, in particular against HIV1MN infection, was associated with reactivity against
the V3MN peptide, while low titre or no reactivity could be detected against the PND from the other strains. This is in agreement with other serological studies which showed the prevalence of V3MN-directed antibodies in m a n y parts of the world (Carrow et al., 1991 ; CheingsongP o p o v e t al., 1992; Warren et al., 1992). React i v i t y was also o b s e r v e d a g a i n s t b o t h the U S / E u r o p e a n and the African consensus V3 peptides, which present 83 and 70 % amino acid homologies with the V3MN peptide, respectively. Nevertheless, the LS children had stronger reactivity against these peptides and significantly higher V3MN IgG avidity than the AIDS children. Low V3IgG avidity could thus be associated with AIDS d e v e l o p m e n t , as has been shown for low p17 IgG avidity (Chargelegue et al., 1993). These data s h o w e d that the MNdirected humoral response can lead to neutralization o f different strains, in agreement with the r e s u l t s o f B e r m a n e t al. ( 1 9 9 2 ) , w h o observed that antibodies to LAI-rgpl20 neutralized only the homologous virus, whereas antibodies to MN-rgpl20 neutralized 6 out of 9 different isolates (Berman et al., 1992). Because syncytium formation is an important step that enables the virus to spread cell-to-cell (Brenner et al., 1991), the fact that the sera inhibited this
100 8O
8O
"~ 60
"~ 60
"= 40 "7.
:~ 40 "7,
20
20
2
3
4 5 6 7 LS children
8
9
10
1 2 3 4 5 6 7 8 9 1011 12 13 14 AIDS children
Fig. 2. Avidity index of the IgG response to HIVluN V3 peptide in sera from LS and AIDS children. The means for the two groups were significantly different (p < 0.05).
A N T I B O D I E S I N R W A N D A N C H I L D R E N W I T H L O N G - T E R M HIV1 I N F E C T I O N
process suggests that the presence of syncytiainhibiting antibodies might be responsible for the slow course of HIV 1 infection in these children. Since the V3 loop was identified as a fusion domain (Freed et al., 1991), essential for gpl20-mediated syncytia formation (Travis et al., 1992), the presence in surviving infected children of high levels of MN-like/PND-specific antibodies supports the notion that the humoral response to the V3 region of HIV1 gpl20 may contribute to protection against disease progression. Indeed, previous studies have described the prevalence of V3-directed antibodies in long-term asymptomatic carriers compared with that of AIDS patients, and have provided evidence for declining levels of these antibodies during the course of infection (Neurath et al., 1990). Moreover, it has recently been reported that the majority of neutralizing antibodies in H I V l - i n f e c t e d p a t i e n t s r e c o g n i z e V3 loop sequences (Vogel et al., 1994). The gpl20 PND lies within a loop formed by a disulphide bridge between two conserved cysteines which play a role in maintaining its tertiary structure. Antibodies directed against this peptide might then alter the protein conformation essential for biological activity (Rossi et al., 1989). Moreover, recent studies indicated that anti-V3 m A b s could neutralize several HIV1 isolates. These broadly neutralizing antibodies were obtained by i m m u n i z a t i o n with HIV1MN V3 peptides (Ohno et al., 1991 ; White-Scharf et al., 1993) and d e f i n e d by their reactivity against the HIV1MN V3 loop (Gorny et aL, 1992). However, none of our sera cross-neutralizing MN and LAI were reactive in ELISA with the V3LAI peptide. As observed by others ( N k e n g a s o n g et al., 1994), our data suggest that there are conserved neutralizing epitopes outside the V3 loop. These epitopes could, in particular, be part of the CD4binding domain of gpl20, whose specific antibodies could neutralize divergent HIV 1 isolates (Steimer et al., 1991). Moreover, a loss of antibodies directed against the CD4-binding site has been associated with rapid disease progression (Cavacini et al., 1993). A previous study of these perinatally HIV 1infected children revealed the presence of p17 IgG and persistent production of IgM to HIV 1
207
envelope glycoproteins, as compared to 16 children with paediatric AIDS (Van de Perre et al., 1992). In the present study, we showed that, despite their small number, sera from these LS children, compared to sera from the AIDS children, had higher neutralizing antibody titres against different HIV1 strains. They also had higher levels of antibodies directed against MNlike V3 peptides, as well as higher V3MN IgG avidity, which could contribute to the prolonged survival of these LS children. We have also observed the presence of non-syncytium-inducing isolates that could be neutralized by the autologous serum, for 4 of the LS children. Delayed HIV1 isolation in culture was also obtained for 2 of them (data not shown). Cell samples were not available for other LS or AIDS children, but these data are in agreement with previous studies showing a correlation between the biological phenotype of HIV1 isolates and the clinical course of infection (De Rossi et al., 1991; Koot et al., 1992; Tersmette et al., 1989). Although sera should be tested for their neutralizing activity against syncytium formarion, as well as against the cytopathic effect induced by a field isolate from Rwanda, our results concerning the humoral response suggest the prevalence of an antigenically MN-related strain in Rwanda, as described in other countries of Africa (Albert et al., 1992; Carrow et al., 1991; Warren et al., 1992). Moreover, because of the strong seroreactivity observed against the A f r i c a n c o n s e n s u s V3 p e p t i d e , we c a n n o t exclude the importance of an African HIV 1 isolate in vaccine development. Nevertheless, the homology of the HIV1MN V3 region with both US/European and African consensus sequences should also be considered in vaccination strategies in this part of Africa.
Acknowledgments This work was supported by the Institut National de la Sant6 et de la Recherce Mrdicale (INSERM) and the Agence Nationale de Recherche sur le SIDA (ANRS), France. C. Fraisier was a recipient from Fondation Marcel Merieux. We wish to thank Drs. V. Boyer and B. Verrier for advice and criticism.
208
C. F R A I S I E R E T AL.
Anticorps dirig6s contre la r6gion V3 proche de la souche MN et neutralisant diff6rentes souches de VIH1, chez des enfants rwandais infect~s par le VIH1 et pr6sentant une survie prolong6e Les s6rums de 11 enfants rwandais infect6s de faqon p6rinatale par le VIH1 et pr6sentant une survie prolong6e (<
References Albert, J., Franzen, L., Jansson, M., Scarlatti, G. Kataaha, P.K., Katabira, E., Mubiro, F., Rydaker, M., Rossi,
P., Pettersson, U. & Wigzell, H. (1992), Ugandan HIV-1 V3 loop sequences closely related to the US/European consensus. Virology, 190, 674-681. Bergeron, L., Sullivan, N. & Sodroski, J. (1992), Target cell-specific determinants of membrane fusion within the human immunodeficiency virus type 1 gpl20 third variable region and gp41 amino terminus. J. Virol., 66, 2389-2397. Berman, P.W., Matthews, T.J., Rddle, L., Champe, M., Hobbs, M.R., Nakamura, G.R., Mercier, J., Eastman, D.J., Lucas, C., Langlois, A.J., Wurm, F.M. & Gregory, T.J. (1992), Neutralization of multiple laboratory and clinical isolates of human immunodeficiency virus type 1 (H1V-1) by antisera raised against gpl20 from the MN isolate of HIV-1. J. Virol., 66, 4464-4469. Blanche, S., Tardieu, M., Duliege, A.M. et al. (1990), Longitudinal study of 94 symptomatic infants with perinatally acquired human immunodeficiency virus infection. Evidence for a bimodal expression of clinical and biological symptoms. A.J.D.C., 144, 12101214. Bowen, D.L., Lane, H.C. & Fauci, A.S. (1985), Immunologic abnormalities in the acquired immunodeficiency syndrome. Annu. Rev. Immunol., 3,477-500. Brenner, T.J., Dahl, K.E., Olson, B., Miller, G. & Andiman, W.A. (1991), Relation between I-~V-1 syncytium inhibition antibodies and clinical outcome in children. Lancet, 337, 1001-1005, Broliden, P.A., Gegerfelt, A., Clapham, P., Rosen, J. Fenyo, E.M., Wahren, B. & Broliden, K. (1992), Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins. Proc. Natl. Acad. Sci. USA., 89, 461-465. Carrow, E.W., Vujcic, L.K., Glass, W.L., Seamon, K.B., Rastogi, S.C., Hendry, R.M., Boulos, R., Nzila, N. & Quinnan Jr, G.V. (1991), High prevalence of antibodies to the V3 region principal neutralizing determinant of HIV-IMN in sera from Africa and the Americas. AIDS. Res. Hum. Retroviruses., 7, 831-838. Cavacini, L.A., Emes, C.L., Power, J., Underdahl, J., Goldstein, R., Mayer, K. & Posner, M.R. (1993), Loss of serum antibodies to a conformational epitope of HIV-1/gpl20 identified by a monoclonal antibody is associated with disease progression. J. Acquired lmune Defic. Syndr., 6, 1093-1102. Chargelegue, D., Colvin, B.T. & O'Toole, C.M. (1993), A 7-year analysis of anti-Gag (p17 and p24) antibodies in HIV-l-seropositive patients with haemophilia: immunoglobulin G titre and avidity are earlier predictors of clinical course. AIDS, 7, $87-$90. Cheingsong-Popov, R., Callow, D., Beddows, S., Shaunak, S., Wasi, C., Kaleebu, P., Giiks, C., Petrascu, I.V., Garaev, M., Watts, D.M., Constantine, N.T. & Weber, J.N. (1992), Geographic diversity of human immunodeficiency virus type 1: serologic reactivity to env epitopes and relationship to neutralization. J. Infect. Dis., 165, 256-261. Chesebro, B., Wehrly, K., Nishio, J. & Perryman, S. (1992), Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unsuai V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism. J. ViroL, 66, 6547-6554.
A N T I B O D I E S I N R W A N D A N C H I L D R E N W I T H L O N G - T E R M HIV1 I N F E C T I O N
De Jong, J.J., Goudsmit, J., Keulen, W., Klaver, B., Krone, W., Tersmette, M. & De Ronde, A. (1992), Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity. J. Virol., 66, 757-765. De Rossi, A., Pasti, M., Mammano, F., Ometto, L., Giaquinto, C. & Chieco-Bianchi, L. (1991), Perinatal infection by human immunodeficiency virus type 1 (HIV-1): relationship between proviral copy number in vivo, viral properties in vitro, and clinical outcome. J. Med. Virol., 35, 283-289. Emini, E.A., Schleif, W.A., Nunberg, J.H., Conley, A.J., Eda, Y., Tokiyoshi, S., Putney, S.D., Matsushita, S., Cobb, K.E., Jette, C.M., Eichberg, J.W. & Murthy, K.K. (1992), Prevention of HIV-1 infection in chimpanzees by gpl20 V3 domain-specific monoclonal antibody. Nature (Lond.), 355, 728-730. Fallon, J., Eddy, J., Wiener, L. & Pizzo, P.A. (1989), Human immunodeficiency virus in children. J. Pediatr., 114, 1-30. Fouchier, R.A., Groenink, M., Kootstra, N.A., Tersmette, M., Huisman, H.G., Miedema, F. & Schuitemaker, H. (1992), Phenotype-associated sequence variation in the third variable domain of human imunodeficiency virus type 1 gpl20 molecule. J. Virol., 66, 3183-3187. Freed, E.O., Myers, D.J. & Risser, R. (1991), Identification of the principal neutralizing determinant of human immunodeficiency virus type 1 as a fusion domain. J. Virol., 65, 190-194. Gomy, M.K., Conley, A.J., Karwowska, S., Buchbinder, A., Xu, J.Y., Emini, E.A., Koenig, S. & Zolla-Pazner, S. (1992), Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. J. Virol., 66, 7538-7542. Hwang, S.S., Boyle, T.J., Lyerly, H.K. & Cullen, B.R. (1991), Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science, 253, 71-74. Javaherian, K., Langlois, A.J., McDanal, C., Ross, K.L., Eclder, L.I., Jellis, C.L., Profy, A.T., Rusche, J.R., Bolognesi, D.P., Putney, S.D. & Matthews, T.J. (1989), Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Natl. Acad. Sci. USA, 86, 6768-6772. Koot, M., Vos, A.H.V., Keet, R.P.M., De Goede, R.E.Y., Dercksen, M.W., Terpstra, F.G., Coutinho, R.A., Miedema, F. & Tersmette, M. (1992), HIV-1 biological phenotype in long-term infected individuals evaluated with an MT2 cocultivation assay. AIDS, 6, 4954. LaRosa, G.J., Davide, J.P., Weinhold, K., Waterbury, J.A., Profy, A.T., Lewis, J.A., Langlois, A.J., Dreesman, G.R., Boswell, R.N., Shadduck, P., Holley, L.H., Karplus, M., Bolognesi, D.P., Matthews, T.J., Emini, E.A. & Putney, S.D. (1990), Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science, 249, 932-935. Lepage, P., Van de Perre, P., van Vliet, G., Nsengumuremyi, F., Van Goethem, C., Kestelyn, P., Msellati, P. & Hitinama, D.G. (1991), Clinical and endocrinologic manifestation in perinatally human immunodeficiency virus type-l-infected children aged 5 years or older. A.J.D.C., 145, 1248-1251. Levy, J.A. (1993), HIV pathogenesis and long-term survival. AIDS, 7, 1401-1410.
209
Myers, G., Berzofsky, J.A., Rabson, A.B., Smith, T.F. & Wong-Staal, F. (1991), Human retroviruses and AIDS, in "Theoretical biology and biophysics". Los Alamos National Laboratory, Los Alamos. Neurath, A.R., Strick, N., Taylor, P., Rubinstein, P. & Stevens, C.E. (1990), Search for epitope-specific antibody responses to human immunodeficiency virus (HIV-1) envelope glycoproteins signifying resistance to disease development. AIDS Res. Hum. Retroviruses., 6, 1183-1193. Nkengasong, J.N., Peeters, M., Ndumbe, P., Janssens, W., Willems, B., Fransen, K., Ngolle, M., Piot, P. & van der Groen, G. (1994), Cross-neutralizing antibodies to HIV-1ArzrT0and H1V-lnm in sera of African and Belgian HIV-l-infected individuals. AIDS, 8, 1089-1096. Ohno, T., Terada, M., Yoneda, Y., Shea, K.W., Chambers, R.F., Stroka, D.M., Nakamura, M. & Kufe, D.W. (1991), A broadly neutralizing monoclonal antibody that recognizes the V3 region of human immunodeficiency virus type 1 glycoprotein gpl20. Proc. Natl. Acad. Sci. USA., 88, 10726-10729. Oxtoby, M.J. (1990), Perinatally acquired HIV infection. Pediatr. Infect. Dis. J., 9, 609-619. Robertson, G.A., Kostek, B.M., Schleif, W.A., Lewis, J.A. & Emini, E.A. (1988), A microtiter cell-culture assay for the determination of anti-human immunodeficiency virus neutralizing antibody activity. J. Virol. Methods., 20, 195-202. Rossi, P., Moschese, V,, Broliden, P.A., Fundaro, C., Quinti, I., Plebani, A., Giaquinto, C., Tovo, P.A., Ljunggren, K., Rosen, J., WigzeU, H., Jondal, M. & Wahren, B. (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. Safrit, J.T., Fung, M.S.C., Andrews, C.A., Braun, D.G., Sun, W.N.C., Chang, T.W. & Koup, R.A. (1993), huPBL-SC1D mice can be protected from HIV-I infection by passive transfer of monoclonal antibody to the principal neutralizing determinant of envelope gpl20. AIDS, 7, 15-21. Steimer, K.S., Scandella, C.J., Skiles, P.V. & Haigwood, N.L. (1991), Neutralization of divergent HIV-I isolates by conformation-dependent human antibodies to gpl20. Science., 254, 105-108. Tersmette, M., Gruters, R.A., De Wolf, F., De Goede, R.E.Y., Lange, J.M.A., Schellekens, P.T.A., Goudsmit, J., Huisman, H.G. & Miedema, F. (1989), Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential isolates. J. Virol., 63, 2118-2125. Tovo, P.A., De Martino, M., Gabiano, C., Cappello, N., D'Elia, R., Loy, A., Plebani, A., Zuccotti, G.V., Dallacasa, P., Ferraris, G., Caselli, D., Fundaro', C., D'Argenio, P., Galli, L., Princip, N., Stegagno, M., Ruga, E., Palomba, E. et al. (1992), Prognostic factors and survival in children with perinatal HIV-I infection. Lancet, 339, 1249-1253. Travis, B.M., Dykcrs, T.I., Hewgill, D., Ledbetter, J., Tsu, T.T., Hu, S.L. & Lewis, J.B. (1992), Functional role of the V3 hypervariable region of HIV-1 gpl60 in the processing of gp160 and in the formation of syncytia in CD4+ cells. Virology, 186, 313-317.
210
C. F R A I S I E R E T AL.
Van de Perre, P., Lepage, P., Simonon, A., Desgranges, C., Hitimana, D.G., Bazubagira, A., Van Goethem, C., Kleinschmidt, A., Bex, F., Brolinden, K., Erfle, V., Gotch, F. & Bumy, A. (1992), Biological markers associated with prolonged survival in African children maternally infected by the human immunodeficiency virus type 1. AIDS. Res. Hum. Retroviruses., 8, 435-442. Vogel, T., Kurth; R. & Norley, S. (1994), The majority of neutralizing abs in HIV-l-infected patients recognize linear V3 loop sequences. Studies using HIV-I(MN) multiple antigenic peptides. J. Immunol., 153, 18951904. Warren, R.Q., Nkya, W.M., Shao, J.F., Anderson, S.A., Wolf, H., Hendrix, C.W., Kanda, P., Wabuke,
M., Boswell, R.N., Redfield, R.R. & Kennedy, R.C. (1992), Comparison of antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gpl60 epitopes in sera from HIV-l-infected individuals from Tanzania and from the United States. J. Clin. Microbiol., 30, 126-131. White-Scharf, M.E., Potts, B.J., Smith, M., Sokolowski, K.A., Rusche, J.R. & Silver, S. (1993), Broadly neutralizing monoclonal antibodies to the V3 region of HIV-1 can be elicited by peptide immunization. Virology, 192, 197-206. World Health Organization. (1986), Acquired immunodeficiency syndrom (AIDS). Weekly Epidemiological Record., 61, 69-73.