Vol. 66, No. 6

JOURNAL OF VIROLOGY, June 1992, p. 3602-3608

0022-538X/92/063602-07$02.00/0 Copyright © 1992, American Society for Microbiology

Cross-Neutralization of Human Immunodeficiency Virus Type 1 and 2 and Simian Immunodeficiency Virus Isolates MARJORIE ROBERT-GUROFF,l* KRISTINE ALDRICH,1 REBECCA MULDOON,' THEODORE L. STERN,1 GEETHA P. BANSAL,2 THOMAS J. MATTHEWS,3 PHILLIP D. MARKHAM,4 ROBERT C. GALLO,' AND GENOVEFFA FRANCHINI' Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20892'; MedImmune, Inc., Gaithersburg, Maryland 208782; Department of Surgery, Duke University Medical Center, Durham, North Carolina 277103; and Advanced BioScience Laboratories, Inc., Kensington, Maryland 208954 Received 12 December 1991/Accepted 16 March 1992

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.

izing a broad spectrum of HIV isolates (38). This property may be due, at least in part, to reactivities directed against the conserved tip of the V3 loop (15). The ability to elicit such a broad immune response has obvious advantages for vaccine development. Previous reports have demonstrated cross-neutralization of HIV-1 and -2 by antibody-positive human sera (2, 3, 39). We show here that cross-neutralization by sera of humans and monkeys infected with HIV-2, SIVMAC, and SIVAGM isolates is more extensive than the infrequent and low-titer cross-neutralizations observed between HIV-1 and -2. Cross-neutralization must reflect conservation of neutralizing epitope. As significant homology exists between the V3 loops of HIV-2 and SIV isolates, we examined the role of the V3 loop of these viruses in neutralization and the immunologic cross-reactivities among them. We show that HIV-2, SIVMAC, and SIVAGM isolates are more similar to each other than to HIV-1 in cross-neutralization properties and in reactivity to complete or partial linear V3 loop peptides. The activity patterns observed reflect the evolutionary relatedness of the envelope genes of these virus isolates (10). In this regard, while the use of linear peptides to elicit neutralizing antibody may be very effective in the HIV-1 system, subunit vaccine approaches in the HIV-2 and SIV macaque models, while similar in theory, may need to differ significantly in character.

The ability of retroviruses to integrate into the host cell genome and establish persistent infection, together with their ability to disrupt normal gene regulation and function, has produced one of the principal challenges in the development of an AIDS vaccine. Attenuated or inactivated preparations of other viral types have proven to be effective vaccines. However, a safer approach for human immunodeficiency virus type 1 (HIV-1) involves identification and use of viral nucleic acid-free immunogenic subunits capable of eliciting protective humoral and cellular immunity. Animal models are crucial for testing putative vaccines. Because of the protected status of primates (chimpanzees and gibbons) susceptible to HIV-1 infection, a sufficient number of animals is not always available for necessary studies. The ability of HIV-2 (7, 21, 26) and simian immunodeficiency virus (SIV) (4, 18) to infect macaques provides alternative primate models for related vaccine development. In order to apply vaccine approaches in the HIV-2 and SIV models to the HIV-1 system, subunit vaccines for these viruses would be useful. One of the principal features of an effective AIDS vaccine will be the ability to elicit neutralizing antibody. The principal neutralizing determinant of HIV-1 has been localized between two cysteine residues in the third variable domain, the V3 loop, of the external envelope glycoprotein, gpl20 (11, 20, 23, 31). While the HIV-1 V3 loop elicits highly type-specific neutralizing antibody, humans naturally infected with the virus develop antibodies capable of neutral*

MATERIALS AND METHODS Sera. Human sera possessing antibodies to HIV-1 or -2 represented individuals both asymptomatic and with AIDS.

Corresponding author. 3602

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TABLE 1. Synthetic peptides representative of HIV-2, SIVMAC, and SIVAGM isolates

Peptide

Virus isolate

Amino acid

1 2 3

HIV-2SBL/ISY

HIV-2SBL,ISY HIV-2SBL,ISY

319-330 307-330 297-330 315-319 330-334 301-324 314-318 332-336

FHSQK(C)

SIVAGMTYO

318-341 316-340

NKTVLPVTIMSGLVFHSQPLTDRP(C)C

HTLV-Y

101-117 319-339

(C)GGPRRSRPRLSSSKDSKf

4

5 6 7 8 9 10

HIV-2SBLISY SIVAGMTYO HIV-2NIH-Z HIV-2N,H-Z SIVMAC251 SIVMAC251 HIV-2SBL/ISY

Sequence

KIINKKPRQAGbC ITLMSGRRFHSQKIINKKPRQAG6C RRPENKTVVPITLMSGRRFHSQKIINKKPRQAGbC

FHSQK(C)

(C)KTVLPITFMSGFKFHSQPVINKKP FHSQP(C)

FHSQP(C)

NKTVLPVTIMAGLVFHSQKYNMKLPd(C) (C)KIINKKPRQAWCRFKGEWREA

Numbering is according to the Los Alamos Database on Human Retroviruses and AIDS. " Glycine is substituted for the tryptophan in the actual sequence. 'Sequence of Franchini et al. (6). "Proline is substituted for the arginine in the actual sequence. HTLV-I, human T-cell lymphotropic virus type I. -f' Sequence of HTLV-I open reading frame II (33).

HIV-1-seropositive samples were obtained from male homosexuals in the United States and Europe. HIV-2-seropositive samples were obtained from Africans or Portuguese who had lived in areas of HIV-2 endemicity, principally GuineaBissau. Sera possessing antibodies to SIVMAC were obtained from three rhesus macaques and one baboon who had been inoculated with SIVMAC251. Sera positive for antibodies to SIVAGM were obtained from African green monkeys naturally infected in Kenya and housed in the United States since 1977. Viruses. Established isolates of HIV-1 (HTLV-IIIB [24], HTLV-IIIRF [24], and RUTZ [38]) and of HIV-2 (NIH-Z [41]) and the infectious molecularly cloned virus, SBL/ISY (5), were prepared as frozen titered stocks. Aliquots of these stocks were used in all assays requiring infectious virus. A similarly prepared SIVAGM isolate, designated 208K, was obtained from fresh peripheral blood mononuclear cells of one of the seropositive African green monkeys by standard techniques. Southern blot analysis established that 208K was a SIVAGM isolate. Peptides. Synthetic peptides (Table 1) were prepared as described previously (14) or by Multiple Peptide Systems (Richmond, Calif.) and used as 80% pure preparations. Peptides conjugated with keyhole limpet hemocyanin were used to immunize mice, guinea pigs, or a goat, following standard protocols. Peptide binding assay. Serum reactivity with synthetic peptides was determined by an enzyme-linked immunosorbent assay (ELISA). Immulon 1 microtiter plates (Dynatech, Chantilly, Va.) were incubated for 1 h at room temperature with poly-L-lysine (50 ,ug/ml, 5 p.g per well) and for 1 h with 0.1% glutaraldehyde. Peptides (20 ,ug/ml, 2 ,ug per well) in carbonate-bicarbonate buffer, pH 9.6, were added and allowed to coat the wells overnight. Following blocking of the wells with 5% bovine serum albumin in carbonate-bicarbonate buffer, 100 pL1 of the antisera at a 1:100 dilution in phosphate-buffered saline (PBS) containing 20% normal goat serum was added, and the mixture was allowed to incubate overnight at 4°C. The plates were washed with PBS-Tween (PBS containing 0.05% Tween 20) and incubated for 1 h with 100 p.l of an appropriate dilution of goat anti-human immunoglobulin G conjugated to horseradish peroxidase per well in PBS-Tween containing 1% normal goat serum. Following

sequential washing with PBS-Tween and PBS, the plates were developed with substrate solution consisting of citratephosphate buffer, pH 5.0, containing 0.005% H202 and 0.05% orthophenylenediamine. The reaction was stopped by the addition of 50 p.l of 4 N H2SO4. A490s were recorded, and the data were expressed as the ratio of the absorbance of the test serum to the absorbance of a standard negative serum. Assay for neutralizing antibody. Neutralizing antibody activity was determined as previously described (28, 29). Amounts of frozen titered stock viruses sufficient to give 50 to 80% infected cells following 5 to 7 days of culture were incubated for 1 h with serial threefold dilutions of serum samples beginning at 1:10. Polybrene-treated target cells (H9 for the HIV-1 isolates, HUT 78 for the HIV-2 and SIV isolates, and MOLT4/clone 8 for the SIVAGM isolate) were added and incubated for 1 h at 37°C. Aliquots of the virus-cell-serum mixture were plated in duplicate into 200 p.1 of culture medium. Virus expression was monitored at appropriate time points for each of the cultures by indirect immunofluorescence assay using a monoclonal antibody for HIV-1, HIV-2, or SIVMAC p24 as appropriate. Virus expression in the SIVAGM assays was monitored by reverse transcriptase assay. Following normalization of the data to infectivity in the presence of a control serum, the neutralization titer was expressed as the reciprocal of the serum dilution at which 60% of the target cells were virus positive by immunofluorescence assay or at which reverse transcriptase levels were 60% of the control value. Assay for peptide inhibition of serum neutralization. The ability of synthetic peptides to inhibit serum neutralization of viral infectivity was assessed by carrying out a standard neutralization assay in which the peptide (0 to 3.6 p.g/rml, final concentration) was incubated with titered stocks of virus and serum prior to the addition of target cells. RESULTS Cross-neutralization of HIV-1, HIV-2, and SIV isolates. Previously, HIV-1 and HIV-2 were shown to cross-neutralize (2, 3, 39). Our experiments confirm a bidirectional cross-neutralization (Table 2). The differences in frequency and titer observed between our data and the earlier reported

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ROBERT-GUROFF ET AL.

TABLE 2. Analysis of cross-neutralizing activity of human and nonhuman primate sera positive for HIV-1, HIV-2, and SIV antibodies Human and nonhuman primate sera antibody positive for:

HIV-2

HIV-1

Virus and isolate

No. of positive Poiie tested samples! no.

(%)

Range of titer

HIV-1 HTLV-IIIB 58/62 (94) 25-2,300 HTLV-IIIRF 35/37 (95) 15->1,250 RUTZ 36/37 (97) 25->1,250 HIV-2 NIH-Z

4/36 (11) 20-110

SIVMACKIW

0/25 (0)

SIVAGM208K

9/24 (38) 10-45

Mean titer

285 >320 >350 40

20

SIVMAC

No. of positive positivef

SIVAGM

No. of

No. of

titer

Mean titer

positive sape/ no. tested (%)

3/8 (38) 1/7 (14) 1/7 (14)

10-30 10 25

15 10 25

0/4 (0) 0/4 (0) 1/4 (25)

75

75

1/5 (20) 1/5 (20) 0/5 (0)

8/8 (100)

65->810

460

4/4 (100)

465->810

590

3/5 (60)

7/8 (88)

25-585

275

4/4 (100)

75

3/4 (75)

sape/ tested tn(%)

1/7 (14)

75

results probably reflect both methodologic details and different demographics of the samples analyzed. No reports of cross-neutralization of HIV and SIV isolates have appeared, except for a single chimpanzee serum possessing anti-HIV-lLAv neutralizing activity which was able to neutralize an SIVSMM isolate at a low titer (8). Here we observed no cross-neutralization of HIV-1 and SIVMAC, except for the serum of one macaque which neutralized the RUTZ isolate, with a modest titer. Cross-neutralization of HIV-1 isolates with SIVAGM-positive sera occurred with a low frequency and a low titer. The reciprocal cross-neutralization of HIV-1-positive sera with the SIVAGM208K isolate was slightly stronger and occurred with greater frequency, similar to that seen with HIV-2-positive sera and HIV-1 isolates (Table 2). In contrast to the weak cross-neutralizations observed between HIV-1 and the other immunodeficiency viruses studied, the cross-neutralization results of HIV-2 and SIV isolates were striking. The majority of serum samples of HIV-2-infected humans, of macaques or a baboon experimentally infected with SIVMAC, and of African green monkeys naturally infected with SIVAGM were able to crossneutralize the HIV-2 and SIV isolates studied, with both a high titer and a high frequency (Table 2). Analysis of the cross-neutralization patterns suggests that HIV-2 is immunologically more closely related to SIVMAC than to SIVAGM, while SIVAGM is more closely related to SIVMAC than to HIV-2. HIV-2 and SIVMAC isolates were cross-neutralized by 88 to 100% of HIV-2- and SIVMAC-positive sera, with titers ranging from 275 to 590, and SIVMAC and SIVAGM isolates were cross-neutralized by 75 to 100% of SIVMACand SIVAGM-positive sera, with titers ranging from 145 to

CRRPENKTVVPI TLM SGRR

Range of ter

Mean ier

positive positiveMea aml/ no. tested

(%)

1,700->8,100 >3,475 30-560

300

5!5

(100)

5/5 (100)

ttr titer

ttr

20 10

20 10

titer

35-135

70

65-405

145

15->810

380

300. The HIV-2- and SIVAGM-positive sera cross-neutralized their respective isolates with lower frequency (14 to 60%) and with only a modest titer (70 to 75). The immunological relationships of these viruses parallel their genetic relatedness. With regard to the envelope gene, SIVAGM is slightly more related to SIVMAC than to HIV-2. Analysis of V3 loop regions. Cross-neutralization reflects conservation of neutralizing epitopes among isolates. The less than 30% homology (Fig. 1) of the V3 loop regions of HIV-1 and HIV-2, represented by the MN, NIH-Z, and SBL/ISY isolates, suggests that an alternate epitope(s) may be responsible for the cross-neutralization observed between these two viral types. The V3 region of HIV-2 has been predicted to form a loop, similar to that of HIV-1 (17). In addition, significant conservation of the V3 region sequence of HIV-2 and SIV isolates occurs at both the amino and carboxy termini of the loops as well as in the central portion (Fig. 1), similar to the homology observed among HIV-1 V3 loops (16). This sequence conservation together with the overall envelope structure of the HIV-2 and SIVs suggests that this V3 region might constitute a neutralizing epitope and also contribute to the cross-neutralization observed. Therefore, we initially examined cross-reactivity patterns of HIV-2- and SIV-positive sera to various V3 loop peptides. Figure 2 illustrates the results of peptide-binding ELISAs of human and nonhuman primate neutralizing sera. The SIVMAC- and SIVAGM-positive sera tested possessed excellent binding reactivities for the homologous and heterologous SIV V3 loop peptides. All were capable of reacting with both of these V3 loops. In addition, the majority of these primate serum samples bound the HIV-2SBL/ISY loop; however, the NIH-Z V3 loop was poorly recognized by the

....

KIINKKPRQAWc

HIV-2S1BLSY

-...................-2NIHZ ...................K -- -

-L...................

SIVMACK SIVAGM O

-T- -NY- - *RKR-H I GP- -A-YTT-N- IGT I - - -H-HIV-1MN FIG. 1. Conservation of V3 loop sequences of HIV-2 and SIV isolates. The V3 loop sequences are aligned and compared with that of the prototype HIV-lMN isolate (9, 12). The boxed area delineates synthetic peptides representative of the various loops. The shaded area denotes the crown region.

VOL. 66, 1992

CROSS-NEUTRALIZATION OF HIV-1, HIV-2, AND SIV

SBLISY

NIH-Z

MAC251

3605

AGMTYO

40r 30k

20k 10

10 11 12 13 14

10 11

--I

SIVAGM Sera 0 co

0 0 -20 en CU

-o(A)

SIVMAC Sera

25r

20k

15k

10o-

1 2 3 4 5

1 2 3 4 5

1

2 3 4 5

1 2 3 4 5

HIV-2 Sera FIG. 2. Binding of HIV-2 and SIV antibody-positive sera to V3 loop peptides in ELISA. Binding is expressed as the ratio of the absorbance of test serum to the absorbance of a standard negative control serum. The dashed lines represent the ratio of 3, below which samples were considered negative. The V3 loops tested, as noted at the top, are those within the boxed area of Fig. 1. *, positive serum sample; 3, negative serum sample.

African green monkey sera. While three of the five HIV-2positive serum samples bound the SBL/ISY V3 loop, and significant cross-reactions of these sera with the SIV peptides were observed, the NIH-Z V3 loop was not recognized by the five HIV-2-positive serum samples depicted, which, nevertheless, were capable of neutralizing the NIH-Z isolate. This fact may suggest that unlike the case with HIV-1, the V3 loop does not play a role in neutralization of HIV-2 infectivity. Alternatively, the binding of HIV-2-neutralizing sera to the V3 loop of HIV-2SBL/ISY and not to that of the NIH-Z isolate may reflect subtle microconformational differences in the peptides studied. The entire loop, minus the amino-terminal cysteine, of the SBL/ISY isolate was represented in the synthetic V3 peptide tested (Table 1, peptide no. 3), while the NIH-Z loop consisted of only the internal 24 amino acids (Table 1, peptide no. 6). In the HIV-1 system, the conserved crown of the V3 loop, the GPGR sequence, plays a major role in immunologic cross-reactivity and neutralization (15). We investigated whether the observed cross-reactivity between HIV-2, SIVMAC251, and SIVAGM2(K could be attributed to the conserved crown region of these viruses.

This putative crown consists of the conserved amino acids FHSQ and either K in HIV-2sBL/Isy and SIVAGM or P in HIV-2N1Hz and SIVMAC (Fig. 1). Comparison of Fig. 2 and 3 shows that while both the SIVAGM- and SIVMAC-positive sera reacted strongly with their homologous V3 loop peptide, as well as with the corresponding heterologous peptide, the SIVAGM-positive sera were much better able to bind their homologous crown peptide. The alteration of a single amino acid in this crown peptide resulted in significantly less binding to the heterologous peptide. In comparison, the SIVMAC-positive sera exhibited good binding to both homologous and heterologous crown peptides. The HIV-2-positive sera, however, were completely unable to recognize either crown peptide. Thus, the conserved central region of the V3 loops of HIV-2 and SIV isolates does not seem responsible for the observed cross-reactive binding activities. Alternatively, the peptides may not have possessed the appropriate conformation for immunologic recognition. Investigation of the V3 loop as a neutralizing epitope. Two recent publications have suggested that the V3 loop of HIV-2 and SIV may function as a neutralizing epitope. In one study, a correlation was observed between cross-neutraliza-

3606

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ROBERT-GUROFF ET AL.

tion and reactivity with the main neutralizing site (3). In a more recent report, carboxy-terminal peptides of the V3 loop of HIV-2 (residues 311 to 330 and 318 to 337) were shown to elicit neutralizing antibodies (1). A peptide similar to the latter (Table 1, peptide no. 10) was not recognized by any of our neutralizing sera, including the HIV-2-positive sera, in binding assays (not shown). Moreover, a goat hyperimmune serum to a peptide similar to peptide no. 10 (R substituted for P at residue 325) and capable of binding peptide no. 10 and the immunogen equivalently was not able to neutralize HIV-2 isolates (not shown). Nevertheless, we further explored the V3 loop as a neutralizing site, making use of hyperimmune sera to V3 loop peptides raised in mice and guinea pigs. Three different strains of mice were immunized with the HIV-2SBL/IsY V3 loop peptide (Table 1, peptide no. 3) and developed peptide-binding antibodies with titers of over 1:10,000 to the immunogen. However, none of these mice developed neutralizing antibody (not shown). Guinea pigs immunized with the HIV-2NIHz V3 loop peptide (Table 1, peptide no. 5) also developed binding antibodies but failed to develop neutralizing antibodies (not shown). We also investigated the ability of V3 loop peptides or V3 loop fragments to inhibit directly serum neutralization. Synthetic peptides no. 1 through 9 (Table 1) were used in competition experiments with HIV-2-positive sera. In addition, the V3 loop peptides of HIV-2sBL/IsY, SIVMAC, and SIVAGM, the SIVMAC crown peptide, and the HTLV control peptide (Table 1, peptides no. 3, 5, 7, 8, and 9) were used in competition experiments of SIVMAC-positive serum neutralization. No consistent competition of serum neutralization of either HIV-2 or SIVMAC was observed. DISCUSSION The ability of HIV-2 and SIV isolates to be cross-neutralized by their respective antisera, together with their poor or nonexistent cross-neutralization of the HIV-1 isolates, was striking. However, a recent analysis of evolutionary relationships suggests that with regard to the env gene, SIVAGM, as well as SIVMND, is more related to the HIV-2 group of viruses, together with SIVMAC and SIVSM, than to the HIV-1 group (10). This relationship is reflected by the extensive homology among the V3 loops of the isolates studied (Fig. 1). Moreover, the envelopes of these viruses possess additional regions of significant homology. Whether such conserved sites as either linear or noncontiguous epitopes can account for the observed cross-neutralizations remains to be determined. This evolutionary grouping of HIV-2, SIVMAC, and SIVAGM distinct from HIV-1 appears to reflect more than envelope relatedness and resulting cross-neutralization properties and extends to identification of the principal neutralizing determinant of the viruses. This determinant for HIV-1 is the V3 loop by virtue of its immunogenicity and its ability to elicit high titers of neutralizing antibodies as well as to absorb neutralizing activity from positive human sera. In contrast, while the V3 regions of HIV-2 and SIV also are immunogenic (22), as shown by the binding reactivity of natural sera, we did not find that they elicit neutralizing antibody or absorb such activity from positive sera. Thus, either this region is not a principal neutralizing site or conformation plays an important role. In this regard, the fact that we did not confirm the results of Bjorling et al. (1) concerning elicitation of neutralizing antibody by synthetic peptides of the HIV-2 V3 loop region may be due to subtle conformational differences in the

5

FHSQP

10_ 5

0 C.)

25 - FHSQK

cr C

m

-20

20k

(A .0

15

10k 5

,,,F///bM777)rM Il

lU I I13Q Z1) 13A4 cD D/ u U HIV-2 Sera Sera SIVMAC SIVAGM Sera FIG. 3. Binding of HIV-2 and SIV antibody-positive sera to the V3 loop crown region. Data are expressed as described in the legend to Fig. 2. 4

peptides used. Our peptide no. 2 (residues 307 to 330, Table 1), which did not absorb neutralizing activity from positive sera, was 4 amino acids longer than one of the Bjorling neutralizing epitopes (residues 311 to 330). Moreover, our peptide no. 10 (residues 319 to 339 [Table 1]), which was not recognized in binding assays by HIV-2-positive sera, was shifted slightly towards the carboxy terminus of the viral envelope compared with Bjorling's second peptide, which

neutralizing antibody (residues 318 to 337). It is also possible that in addition to conformational changes caused by slight differences in primary amino acid sequences, derivatized by-products of the various peptide syntheses

elicited

may influence conformation and resultant immune recognition and function. Such effects have been described for CD4 peptide fragments which inhibit HIV infection (19) and may also contribute to the different results reported here. One can infer conformational influences from the peptidebinding studies. The SIVAGM-positive sera cross-reacted with both the SIVMAC V3 loop and the HIV-2SBL/lSy V3 loop peptides in which the central 25 amino acids are represented (Fig. 1 and 2). However, very poor binding to the NIH-Z V3 loop peptide, which shares considerable homology with the other V3 loops (Fig. 1), was observed. Conformational differences in this peptide may explain the data. The SIVMAC-positive sera exhibited the greatest crossreactivity, presumably directed in part to the crown region. They possessed reactivity to both linear crown peptides (Fig. 3) in contrast to the African green monkey sera with which a single amino acid substitution abolished binding activity in all but one of the serum samples (Fig. 3). The binding properties of the HIV-2-positive sera are of greater interest because of the fact that the sera studied were all capable of neutralizing the NIH-Z isolate yet did not bind the NIH-Z V3 peptide. On this basis, one might conclude that the V3 loop is not important for neutralization of HIV-2. Three of five of these serum samples, however, bind the

VOL. 66, 1992

CROSS-NEUTRALIZATION OF HIV-1, HIV-2, AND SIV

SBL/ISY V3 loop, suggesting that the longer length of this peptide may provide requisite conformational epitopes for immunologic recognition. Resolution of the role of the V3 loop is complicated by the fact that the SBL/ISY cloned virus is not neutralized by these sera and, in general, is difficult to neutralize. Further studies with the parental SBL/6669 strain will address this point. Finally, the lack of binding to the NIH-Z V3 loop may simply reflect type specificity of the sera studied. Such differential binding by broadly neutralizing sera has been described for HIV-1 (25). While the primate sera studied were able to bind at least their homologous crown peptide (Fig. 3), the HIV-2-positive sera could not recognize these linear peptides at all. Moreover, the crown peptides were not able to absorb neutralizing activity from either the HIV-2- or SIVMAC-positive sera (data not shown). Thus, these studies suggest that in contrast to the HIV-1 system, this central V3 region, although highly conserved, is not responsible for neutralization of HIV-2 or SIV in a linear format. Although the number of serum samples studied was small, the level of cross-neutralization observed between HIV-2 and SIV isolates suggests a structural and functional similarity of these viruses and sets them apart from HIV-1. Certainly, neutralization of these viruses does not simply occur as an immune response to the linear V3 loop as a principal neutralizing epitope. However, in view of the similarity in overall features of the HIV-1 and -2 envelopes (17) and the conserved nature of the V3 loops, it is difficult to imagine that the V3 region of HIV-2 and SIV does not play some major role in the infectivity or biologic functioning of these viruses as it does in HIV-1. Moreover, it would be surprising if HIV-1 did not also possess conformational determinants. In fact, previous studies have clearly shown that the HIV-1 V3 loop is not the only neutralizing determinant of the virus and probably does not elicit the broadly reactive neutralizing antibody activity observed for naturally infected individuals (25, 35). Moreover, several lines of evidence have pointed to a role of conformational epitopes in HIV-1 neutralization. A single amino acid substitution outside the V3 loop of an HIV-1 escape mutant immune selected in vitro (30) was shown to result in neutralization resistance (27), apparently due to conformational changes (40). Broadly reactive, conformation-dependent neutralizing antibodies which do not recognize the V3 loop have been purified from HIV-1-positive human sera by affinity chromatography on native, glycosylated, denatured envelope protein (36). In addition, characterization of a broadly reactive human neutralizing monoclonal antibody has suggested that it recognizes four noncontiguous sites on the viral envelope (37). In view of these findings, although HIV-1 isolates may differ from the HIV-2 and SIV group of isolates, both groups may share important structural, biological, and immunological features. It seems clear that as models for HIV-1 subunit vaccine development, HIV-2 and SIV may be theoretically suitable. However, the precise subunits used in these cases may not be able to be restricted to synthetic peptides. Native proteins, completely and naturally glycosylated, may be necessary. On the other hand, the partial protection of monkeys from an SIV challenge conferred by alternate peptide subunits (34) suggests that linear epitopes may well play a significant role in future vaccine preparations. In addition, the possibility should not be overlooked that HIV-2 and SIV may infect and be neutralized somewhat differently from HIV-1. A recent report indicates that HIV-1 isolates can only partially interfere with superinfection by HIV-2, indi-

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cating that inherent differences in receptor interactions may exist between the two viruses (13). Furthermore, while HIV-1 and HIV-2 both utilize CD4 as a receptor (32), the different host ranges of the viruses suggest that secondary receptors may play a role in infectivity. In studying HIV-2 and SIV macaque vaccine models, we should thus not overlook quite distinct noncontiguous epitopes and the possibility of synergistic neutralizing antibodies. Study of alternate neutralizing sites and conformational determinants may help elucidate mechanisms of infection of all the human and subhuman primate lentiviruses. ACKNOVVLEDGMENTS We thank Ermalinda Cardoso for providing the human HIV-2positive sera; Phyllis Kanki for the SIV antibody-positive rhesus macaque and baboon sera; and James Goedert, William Blattner, and Otto Thraenhart for the HIV-1-positive human sera.

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