Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)

0161-5890/90 $3.00 + 0.00 cj I990 Pergamon Press plc

Moleculur Immunology, Vol. 27, No. 6, pp. 539-549, 1990 Printed in Great Britain.

CONFRONTING THE HYPERVARIABILITY OF AN IMMUNODOMINANT EPITOPE ELICITING VIRUS NEUTRALIZING ANTIBODIES FROM THE ENVELOPE GLYCOPROTEIN OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-l) A. The Lindsley

F. Kimball

Research

R. NEURATH

Institute,

and N.

STRICK

The New York Blood Center, NY 10021, U.S.A.

310 E. 67th Street, New York,

(First received 23 October 1989; accepted 18 December 1989) Abstract-Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-l) are considered importaqt for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-l gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-l are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-l. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-l isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-I IIIB. The results suggest the possibility of developing broadly protective HIV-l immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.

INTRODUCTION

Systematic

B-cell

glycoproteins thetic

peptides

epitope

gp120

and

revealed

mapping gp41 the

of the

of HIV-l

location

from the hypervariable V3 region of HIV-l gp120 (Modrow et al., 1987). Therefore, synthetic peptides from this region elicit virus neutralizing antibodies which appear to be strictly HIV-l subtype (isolate)specific (Javaherian et al., 1989; Palker et al., 1989). HIV-l undergoes sequence variations during in vivo and in vitro replication and HIV-l isolates cannot be described in defined molecular terms and should be considered as quasi-species (Goodenow et al., 1989; Meyerhans et al., 1989). The high sequence diversity of HIV-l proteins is likely to have a considerable impact on the efficiency of immunological surveillance against HIV-l infection. The obvious contradiction between the proposal that only conserved regions should be considered for the development of general strategies to control HIV-l infection (Goodenow et al., 1989) and the experimental findings indicating that hypervariable regions are the most effective in eliciting virus neutralizing antibodies have to be reconciled. One way to accomplish this goal consists in understanding the limits of hypervariability within immunodominant epitopes important for virus neutralization and in defining the extent of immunological cross-reactivities between analogous virus neutralizing epitopes from distinct, already sequenced HIV-l isolates and clones. Steps towards accomplishment of this goal are reported here.

envelope using

syn-

of a dominant

eliciting virus neutralizing antibodies within the sequence (306338) of HIV-l (clone BH-10) gp120 (Neurath et al., 1990). This corresponds to the sequence 443 in the top line of Fig. 1. Shorter peptides from this region also elicited virus neutralizing antibodies (Goudsmit, 1988; Goudsmit et al., 1988; Matsushita et al., 1988; Palker et al., 1988; Rusche et al., 1988; Javaherian et al., 1989). Peptide (306338) from HIV-l BH-10 gp120 was highly immunogenic in free form without attachment to a protein carrier (Neurath et al., 1990), indicating that it contained both B- and T-cell epitopes. The presence of T-cell epitopes in this region of HIV-l gpl20 was also directly demonstrated using synthetic peptides (Takahashi et al., 1988; Palker et al., 1989). Therefore, peptides from this region of HIV-l gp120 may be considered as candidate components for future vaccines for prevention of acquired immunodeficiency (AIDS). The peptide (306338) from HIV-l BH-10 (Neurath et al., 1990), as well as similar shorter peptides described by others (see above) are derived epitope

539

540

A. R. NEURATH and N. STRPZK

BH-10 g;:: PV-22 :: g-2

..

E-5 gj::

ml-3 CD 451 K” JY-1 :-:” ::: JH3

Fig. I. Amino acid sequences corresponding to hypervariable loops from the V3 region of 22 HIV- 1 isolates (bottom). For maximum sequence alignment, gaps were introduced into some of the sequences. Conserved sequences are boxed. Variability of the amino acid sequence corresponding to the hypervariable loops from 22 HIV-I isolates (top). Variability analysis was done according to Wu and Kabat (1970) (B) and a divergence score method described in the text (m).

MATERIALS

AND METHODS

Pep tide synthesis Peptides from the V3 hypervariable loop of gpl20 of distinct HIV-I isolates listed in the bottom part of Fig. 1 (except the peptide from the JH3 sequence; Komiyama et al., 1989) were synthesized. The sequences are derived from Myers et al., 1988 and Anand et al., 1989. The peptides were obtained from two sources: Neosystem Laboratories, Strasbourg, France (N; from HXB-2, HXB-3, PV-22, MN, WMJ-3, SF-2, BR, MAL and Z-3 isolates) and Dr R. Fields, The New York Blood Center (F; from BH-10, RF, SC, WMJ-1, WMJ-2, NY-5, CD-451, JY-1, ELI, Z-6, LAV-MA and Z-321 isolates). C-terminal tyrosines (Y) were added to peptides lacking Y residues in their sequence (BH-10, HXB-2, HXB-3, PV-22 and Z-321 isolates). Peptides from the first source (N) were synthesized as follows: Boc-amino acids were purchased from Propeptide (France). Both Boc-Cys (4-MeBzl) PAM resin and Boc-Glu (OcHx) PAM resin were prepared as described (Plaue and Heissler, 1987). For trifunctional amino acids, the following side chain protecting groups were used: Tosyl for Arg, cyclohexyl for Asp and Glu, 4-methylbenzyl for Cys, benzyl for Thr,

chlorobenzyloxycarbonyl for Lys, 2,6-dichlorobenzyl for Tyr, formyl for Trp, benzyloxymethyl for His and sulfoxide for Met. All syntheses were carried out on an NPS 4000 semi-automated multichannel peptide synthesizer. All coupling reactions were performed in dimethylformamide with a 3-fold excess of hydroxybenzotriazol active esters. The reactions were continued until no free amines could be detected by the qualitative ninhydrine test. For cleavage of peptides from the resin, the classical low-high HF procedure was used. To avoid polymerization due to Cys residues, the crude product was directly purified (without lyophilization) by medium pressure liquid chromatography (MPLC). The purity of the product was confirmed by HPLC and by amino acid analysis. All synthesis and purification procedures are described in more detail elsewhere (Plaue and Heissler. 1987; Ven Regenmortel et al., 1988). Peptides from the second source (F) were synthesized on the Biosearch Model 9600 automatic peptide synthesizer using benzotriazolyloxytris (dimethylamino)-phosphonium hexafluorophosphate (BOP) plus I-hydroxybenzotriazole (HOBt) activation in a 9-fluorenylmethoxycarbonyl (Fmoc)-mediated synthesis (Biosearch Technical Bulletin No. 9000-03, Biosearch Inc., San Rafael, California). The sequence

541

Synthetic peptides from HIV-l of each peptide was determined using an Applied Biosystems Protein Sequencer Model 477A with online HPLC analysis of PTH amino acids using an Applied Biosystems Model 120A PTH analyzer. Immunization

of rabbits

Two NZW rabbits were immunized with 200 pg of the respective peptides in combination with complete Freund’s adjuvant. Before use for immunization or for radioimmunoassays the peptides were oxidized by exposure to air in phosphate buffered saline (0.01 M phosphate pH 8, 0.14 M NaCl) overnight to allow the formation of disulfide bonds between the N- and C-terminal cysteines. The rabbits were boosted with 2OOkg doses of peptides in combination with incomplete Freund’s adjuvant in biweekly intervals. Two weeks after each immunization, blood samples were taken and analyzed for antibodies by radioimmunoassay (RIA). Ten weeks after the initial immunization, the rabbits were sacrificed after collecting blood by cardiac puncture. Rabbits (2) were also immunized with a mixture of all 21 peptides synthesized (10 pg of each peptide per dose = a total of 2lOpg of distinct peptides per dose). Double-antibody

radioimmunoassays

(RIA)

Wells of 96-well polystyrene plates (Immulon II, Dynatech Laboratories, Inc., Chantilly, Virginia) were coated with the respective synthetic peptides (200 111;20 pg/ml in 0.1 M Tris, pH 8.8) overnight at 2O’C. The wells were post-coated with bovine serum albumin (BSA) and gelatin (10 and 2.5 mg/ml, respectively). To determine the immune response of rabbits immunized with the mixture of 21 peptides from the hypervariable loop of distinct HIV-l isolates, wells coated with a mixture of all 21 peptides (equal quantities of each peptide; total concentration of peptides = 20 /*g/ml) were used. Wells of 96-well plates coated with recombinant gpl60 (500 ng/ml; MicroGeneSys, Inc., West Haven, Connecticut) were used to detect antibodies recognizing HIV-l gp120. Control as well as anti-peptide antisera were serially diluted (starting dilution exceeding 1: 20) in a mixture of fetal bovine serum and goat serum 9: 1, 0.1% Tween 20, adjusted to pH 8.0. The quantity of attached rabbit IgG was determined from the subsequent attachment of ‘?‘I-labeled second antibodies as described before (Neurath et al., 1987). Dilution endpoints were calculated as described by Ritchie et al., 1983. Geometric mean endpoints corresponding to 2 antisera per peptide are presented in the Results section. The individual endpoints differed by
(isolate IIIB) proteins. These strips were from a commercial test kit for detection of anti-HIV (DuPont, Wilmington, Delaware). The attachment of rabbit antibodies to the separated HIV-l gpl20 or gp41 was detected by biotinylated goat anti-rabbit IgG antibodies from Bethesda Research Laboratories (Gaithersburg, Maryland) followed by avidin-conjugated horseradish peroxidase from the HIV Du Pont kit. Procedures recommended by the manufacturer of the kit were followed. Pooled human anti-HIV-l was assayed at a dilution of 1:lOO. Virus-neutralization

assays

Anti-peptide antisera obtained after final bleedings of rabbits, as well as sera collected before immunization, used as controls, were tested. Immunoglobulins were precipitated by ammonium sulfate (40% saturation), redissolved in a volume of 0.14 M NaCl; 0.01 M Tris pH 7.2 (TS) corresponding to the original volume of sera, dialyzed against TS and filtered through 0.45 pm pore-size filters. The titer of virusneutralizing antibodies was determined by two distinct methods, based on inhibition of synthesis of the core protein P24 and on protection by antibodies of cells against the cytopathic effect of HIV-l (colorometric method), respectively. Geometric mean titers corresponding to 2 antisera per peptide are presented in the Results. For the first assay, samples were serially diluted in RPM1 1640 medium containing 10% fetal calf serum (FCS) and 1% glutamine. Each dilution was filtered through 0.2pm Centrex cellulose acetate discs. Filtered aliquots were added to wells of 96-well plates and mixed with an equal volume of diluted HIV-l [multiplicity of infection (MOI) = 0.0031. After incubation for 1 hr at 37°C MT-2 cells (Harada et al., 1985) were added to each well. After incubation for 1 hr at 37°C the medium was removed and replaced by fresh medium. After incubation for 45 days at 37°C the medium from each well was assayed for P24 using a kit from Coulter Immunology, Hialeah, Florida. For the calorimetric assay, diluted, filtered samples in 96-well plates were mixed with an equal volume of HIV-I-BH-10, added to a final MO1 of 0.0045. The plates were incubated for 60 min at 37°C 25 ~1 of polybrene-( 1 pgg/ml)-treated MT-2 cells (5000 cells/well) were added. The mixture was incubated for 60 min at 37°C and the volume was adjusted with RPM1 1640 medium with 10% FCS to 200~1. After incubation at 37°C for 5 days, an indicator XTT tetrazolium salt (PolySciences, Inc., Warrington, Pennsylvania) was added. After 4 hr, intracellular formazan formation was determined colorometrically. The titers of virus-neutralizing antibodies in human anti-HIV-l positive sera measured by either of the two assays ranged between 1: < 10 and I :200 (A. Hellman and A. K. Fowler, personal communication).

542 Variability

A. R. NEURATH~~~ anaIysis

The divergence between sequences of peptides listed in the bottom part of Fig. 1 was determined by two distinct methods: (a) the variability analysis described by Wu and Kabat (1970) and (b) by calculating divergence scores between pairs of peptides using a scoring matrix described recently (Risler et al., 1988). The divergence scores for each of the amino acid residues from all possible comparisons 23 1 = 2 I + 20 + 19 + etc.) of pairs of sequences listed in the bottom part of Fig. 1 were summed and divided by the number of all possible comparisons (231). The resulting number was multiplied by the variability calculated according to Wu and Kabat (1970). The cross-reactivity between distinct peptides was calculated in percentages as follows:

% cross-reactivity

=

N.STRICK

whether inclusion of these new results will affect the variability plots, which in the presented form indicate the highest variability at amino acid residues 16, 30, 37, 1 I, 14, 35, 25, 15 and 38 (listed according to decreasing variability scores). Our earlier studies (Neurath et al., 1990) indicated that synthetic peptides corresponding to hypervariable loops of 21 distinct HIV-1 isolates (sequences listed in Fig. 1, except the sequence corresponding to the JH-3 isolate) were recognized by antibodies in sera of HIV-I infected individuals and by an antiserum to HIV-l (isolate IIIB) raised in rabbits. This suggested that at least some of the sequences listed in Fig. 1 are immunologically cross-reactive despite their sequence divergence. To explore this issue in great detail, antisera to the distinct hypervariable

reciprocal peptide

of dilution endpoint corresponding to the reaction with heterologous antipeptide antiserum

of

reciprocal peptide

of dilution endpoint corresponding to the reaction with homologous antipeptide antiserum

of

All unidirectional cross-reactivities were calculated in this way. The bidirectional cross-reactivity between peptides (A and B) was calculated as follows: Bidirectional cross-reactivity = geometric mean of the immunological cross-reactivity calculated for the reaction between peptide A and antibodies to peptide B and of the immunological cross-reactivity calculated for the reaction between peptide B and antibodies to peptide A.

RESULTS

Variability of sequences corresponding to hyper variable loops .from the V3 region of distinct HIV-l isolates The comparison of published sequences corresponding to hypervariable loops from the V3 region of distinct HIV-1 isolates indicates that these sequences are highly variable, only 8 amino acid residues being conserved among 22 distinct sequences (Fig. 1). The maximum alignment of these sequences was possible only by introducing gaps (Fig. 1, top). The variability among these sequences was analyzed by variability plots (Wu and Kabat, 1970) and found to be generally less extensive than that observed for immunoglobulin variable regions (Kabat et al., 1987). To distinguish between conservative and nonconservative amino acid replacements, the variability plot was modified by assigning distinct scores to different amino acid replacements (Risler et al., 1988). The corresponding results are also plotted in the top part of Fig. 1. The sequence of hypervariable loops from 138 HIV-l isolates has been reported recently (LaRosa er al., 1990). It is not known

loop peptides were prepared and used to study the serological cross-reactivity between the peptides. Immunogenicity of synthetic peptides .from the hypervariable loop of distinct HIV-l isolates Antisera to each of the 21 peptides corresponding to sequences listed in Fig. 1 (bottom; except sequence JH-3) as well as antisera to a mixture containing equal amounts of each of these peptides were raised in rabbits in order to answer the following questions: (1) are the peptides immunogenic and do amino acid replacements affect their immunogenicity; (2) do the antipeptide antibodies recognize epitopes on the native HIV-I envelope glycoprotein; (3) is it possible to prepare antisera recognizing a whole array of HIV-l glycoproteins differing in amino acid sequence of their hypervariable loops by immunization with a mixture of peptides derived from the corresponding highly divergent sequences; and (4) are antibodies elicited by full length synthetic loop peptides from distinct HIV-l isolates virus neutralizing? Each of the 21 peptides, without conjugation to a protein carrier, elicited high levels of antibodies to the respective homologous peptides (dilution endpoints exceeding 1: 105; Fig. 2, dashed columns). Antisera obtained by immunization with a mixture of all 21 peptides elicited antibody responses to each of the peptides (Fig. 2, black columns). The endpoint dilution titers as measured by RIA with individual peptides exceeded 1: 50,000, except for antibodies to peptides from the LAV-MA and JY-1 isolates. Thus, by combination of an array of peptides into a single immunogen, it is possible to overcome problems arising from the limited cross-reactivity between hypervariable loops from the V3 region of distinct HIV-I isolates (see below).

Synthetic

peptides

q

Antiserum

against homologous

m

Antiserum

against

543

peptide

mixed peptide

SYNTHETIC

from HIV- 1

(21)

PEPTIDES

Fig. 2. Immunogenicity of peptides derived from the V3 hypervariable loop of distinct HIV-I isolates, Rabbit antisera to the individual peptides (two rabbits per peptide) were raised and tested by RIA on wells coated with the respective homologous peptides (B). A pool of 21 peptides from distinct HIV-I isolates was also used to immunize rabbits and the resulting antisera were assayed by RIA on peptides from distinct HIV-l isolates (W). Rabbits received five doses of individual peptides (200 fig) in combination with complete and incomplete Freunds adjuvant. The peptide mixture consisted of 10 pg of each peptide (total = 210 pg).

To assess the cross-reactivity between epitopes on the respective synthetic peptides and on HIV-I envelope glycoproteins, the reactivity of the antisera to synthetic peptides with recombinant gpl60 (rgp160) was studied. These investigations had to be limited to rgp160 from the IIIB isolate, since similar products derived from other HIV-l isolates (clones) are not commercially available. The dilution endpoint of antibodies recognizing rgp160 in the antiserum raised against the BH-10 peptide was 1:203,000, similar to the dilution endpoint of antibodies recognizing the homologous peptide (1: 243,000). These results indicate a considerable similarity of epitopes exposed on the viral glycoprotein and on the synthetic peptides, and the absence of a dominant population of antipeptide antibodies not recognizing the native protein in the antipeptide antiserum. Other antipeptide antisera, except anti-MAL and anti-ELI, also reacted with rgpl60-IIIB (Fig. 3, black columns). Cross-reactivity > 10% was seen only with antisera to peptides from the HXB-2, HXB-3, PV-22, SC, RF, SF-2 and MN isolates. For comparison, the reactivity of the distinct antipeptide antisera with the BH-10 peptide was also studied (Fig. 3, dashed columns). Reactivity of these antipeptide antisera with the BH-10 peptide was greater than that with rgpl60-IIIB. The antiserum raised against a mixture of 21 peptides contained antibodies recognizing rgpl60IIIB (Fig. 3; endpoint titer 1:25,000) and antibodies recognizing the BH-10 peptide (endpoint titer 1: 321,000). This difference in titers may possibly be ascribed to a more extensive cross-reactivity between peptides from distinct HIV-l isolates as compared

with the cross-reactivity of the respective peptides with rgpl60-IIIB, as is also indicated by the distinct lengths of dashed and black columns in Fig. 3. All antipeptide antisera were also evaluated by Western immunoblots containing separated polypeptides from HIV-I-IIIB. Only antisera to peptides from the BH-10, HXB-2, HXB-3, PV-22, SC, RF, SF-2 and WMJ-3 isolates reacted (Fig. 4). Antibodies in the antiserum raised against a mixture of the 21 hypervariable loop peptides, as expected, also recognized gpl20-IIIB in Western blots (right lane; Fig. 4). Antipeptide antisera displaying a cross-reactivity with rgpl60-IIIB 2 5% (Fig. 3) were evaluated by virus neutralization assays. These studies were limited to the HIV-I-IIIB isolate, routinely used in virus neutralization assays. Virus neutralizing activity was consistently detected only in antisera to peptides corresponding to the BH-10, HXB-2, HXB-3, PV-22 and RF isolates (Table 1). These results suggest that immunological cross-reactivity measured by virus neutralization is more profoundly affected by amino acid replacements in the sequence of the V3 hypervariable loop than is immunological cross-reactivity measured by serological reactions. Immunological cross-reactivity between peptides derived from the hypervariable loop of the V3 region ofgp 120 from distinct HIV-l isolates (clones) In addition to the specificity of virus neutralizing antibodies, the specificity of other antibodyand cell-mediated immune responses [antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent complement mediated cytotoxicity (ACC) and

A. R. NEURATH~~~

N.STRICK

m

ANTI-PEPTIDE

BH-IO

Peptide

fl

ANTISERA

Fig. 3. Recognition of recombinant gp160 (m) and the peptide (303-338) (S) from the BH-10 isolate by antibodies to synthetic peptides from the V3 hypervariable loop (Modrow et al., 1987) of distinct HIV-I isolates. cytotoxicity mediated by cytotoxic T-lymphocytes (CTL)] is affected by amino acid replacements within the protein target sequence. Considering the large number of HIV-l isolates differing in primary sequence, studies on the effect of amino acid replacements on the specificity of these immunological reactions, contributing to protection against disease,

requires a major effort. Knowledge concerning serological cross-reactivities between hypervariable loops from the V3 region of distinct HIV-I isolates would provide useful background for such extensive studies. Towards this goal, the serological crossreactivities between peptides from the hypervariable loop of 21 HIV-l isolates (clones) were studied.

Fig. 4. Western immunoblots with antipeptide antisera. For comparison, an immunoblot obtained with a pool of sera from HIV-I infected individuals is shown on the left. Immunoblots obtained with sera from rabbits before (pre) and after (post) immunization with peptides are shown.

Synthetic Table 1. Neutmiization peptides

of HIV-I-IIIB from distinct HIV-l

infectivity by antisera isolates (clones)

peptides to

Virus-neutralization assay results (endpoint dilution) by Aotipept~de antiserum

P24 detection

Anti-BH IO Anti-HXB-2 AM-HXB-3 Anti-PV-22 Anti-SC Anti-RF Anti-SF-2 Anti-21 loop peptidr mixture

I:8 I:100 I:16 1:lMt <1:5 I:25
Calorimetric method I:12 1:124 I:85 1:560 1:lO I:8 I:14 1:80

Results of these studies are summarized in Table 2 and indicate extensive cross-reactivities between a few sequences and much more limited erossreactivities among most of the 21 peptides. Cross-reactivities exceeding 25% are presented in Table 3. Considering the great divergence among amino acid sequences among V3 hypervariable loops, it Table 2. Reaction

from HIV-l

545

would be of value to be able to predict the extent of immunological cross-reactivity from the amino acid divergence between the distinct peptide sequences. For this reason, it was necessary to express the divergence between these sequences in a quantitative fashion. To accomplish this, the divergence scores for pairs of peptides from the V3 hypervariable loops were calculated and summarized in Table 4. From this table, it is possible to identify pairs of sequences expected to be the most related. By combining data presented in Tables 2 and 4, it was possible to relate serological cross-reactivity to amino acid divergence. The corresponding relationship approximates a linear regression with a correlation coefficient r = 0.718 (Fig. 5). The dilution endpoint of the antiserum to the MN peptide with either the homologous peptide or the peptide derived from the NY-5 sequence was not affected by a short pcptide IRQAHC (common for the N-terminus of the MN and NY-5 sequence; Fig. I), added to a final concentration of 50 pg/ml during incubation of serial dilutions of the antiserum with wells coated either with the MN or NY-5

of antisera lo each of the 21 peptides from ihe hypervariable of distinct HIV-I isolates with homologous and heterologous

loops from the V3 region peptides

Each antiserum was tested on wells of polystyrene plates coated with individual peptides. Dilutions of each antiserum were assayed by double antibody RIA and dilution endpoints were calculated. The dilution endpoint for the reaction between each antiserum and the homologous peptide was considered as 100%. Percentage of cross-reactivity was determined by dividing the reciprocals of the dilution endpoints for the respective heterologous reaction with those corresponding to the reaction between homologous reactants. Cross-reactivities > 25% are emphasized by boxes.

A. R. NEURATH and N. STRKK

546

Table 3. Compilation of selected results from studies concerning the immunologic cross-reactiwty between peptides from distinct HIV-isoktes Ant;serum

.Anti-BHIO

Anti-HXB-2 Anti-HXB-3 Anti-PV-22 Anti-SC Anti-RF Anti-SF2 Anti-MN Anti-BR Anti-NY-5 Anti-WMJ-I Anti-WMJ-2 Anti-WMJ-3 Anti-CD451 Anti-LAV-MA Anti-Z-3 Anti-JY-I Anti-Z-321 Anti-Z-6 AntI-MAL Anti-EL.1

Peptides recognized (Cross-reactivity 2 25%) BHIO, HXB-2, HXB-3, PV-22, RF. NY-5 ---BHIO, HXB-2, HXB-3, PV-22 BHIO. HXB-2, HXB-3, PV-22, SF-2 BHIO, HXB-2. HXB-3, PV-22, SF-2 SC, Z-321 RF SC, RF, SF-2, BR, NY-5, WMJ-3, Z-321 MN RF, BR SC. RF, NY-5 SC, RF, WMJ-I, CD451 RF. WMJ-2

BHiO, NY-5, WMJ-I, WMJ-3, CD451 CD451 RF LAV-MA z-3 SC, RF, SF-2, BR, NY-5, JY-l,Z-32l.Z-6, SC, WMJ-I, WMJ-2, Z-321 Z-6 NY-5 MAL ELI

DISCUSSION

ELI

peptides. Similarly, the dilution endpoint of antibodies to the RF peptide with either the homologous peptide or the HXB-2 peptide was not effected by the presence of the peptide CTRPNNNTRK from the N-terminus of both the RF and HXB-2 sequence (Fig. 1) (data not shown). These results suggest that the observed serological cross-reactivity between distinct HIV-I peptides listed in Fig. 1 is either mostly due to sequences representing the central portion of the hypervariablc loop or that the short C- and N-terminal peptides differ in their respective conformations sutliciently from the conformations of the corresponding regions in the full length loop peptides, so that they do not affect the outcome of serological reactions described above. The immunological cross-reactivity between some of the V3 hypervariable loop peptides suggests the possibility of preparing antisera recognizing an array Table 4. Divergence

scores calculated

for pairs of peptides

of HIV-I isolates by immunizing with peptides derived from selected sequences rather than from ali sequences from these isolates (Tables 1. 3; Fig. 5). The possibility to forecast serological crossreactivities between distinct peptides from amino acid sequence divergence scores may help to design immunogens consisting of a series of synthetic peptides collectively eliciting a broad immune response to already identified HIV-I isolates and to additional variants arising in the course of selection and mutagenesis occurring during in zGco HIV-I replication.

Virus neutralizing antibodies against HIV- 1 are predominantly subtype specific and directed against epitopes on a hypervariable loop from the V3 region of the envelope glycoprotein gp120 (Javaherian et al., 1989; Neurath ef al., 1990). Epitopes recognized by helper and cytotoxic T-cells are also located on this hypervariable loop. Therefore, T-helper cell responses to HIV-l gp120 as well as cell-mediated cytotoxic responses are expected to be also subtype specific. The observation that protective immune responses are directed to highly variable regions of virus surface proteins is not unique for HIV-l. Analogous findings apply to other viruses, including for example influenza virus (Barnett et al., 1989; Graham et al., 1989). Unlike antigenic shifts and drifts observed with influenza viruses within relatively long time spans, HIV-I subtypes with extensive differences in primary sequence of envelope glycoproteins circulate simultaneously in the infected population and new subtypes are generated as a result of selection and mutagenesis in a single infected individual (Goodenow etal.,1989:Meyerhans er ul., 1989). For this reason, vaccines against HIV-I have to be effective simultaneously against an array of

from the V3 hypervariable loop of HIV-I the recently sequenced JH-3 strain)

isolates enumerated

in Fig.

I (except

Divergence scores were calculated by summing scores for each amino acid replacement within the sequence using a recently publish~ scoring matrix (Fig. 5 in Risk ef al., 1988), and by adding 2.2 for each amino acid residue represented in one but not in the paired sequence and 6.0 for each gap in the sequence (Dayhoff ef ul., 1983). Divergence scores Q II are boxed to emphasize predicted significant cross-reactivities larger than 10% (Fig. 5).

Synthetic virus

isolates.

To generate

such

broadly

peptides

effective

in great detail the immunological cross-reactivity between protective epitopes on HIV-l envelope glycoproteins and the relationship between immunological specificity and primary sequence divergence. In the first step towards accomplishment of this goal, the serological cross-reactivity between synthetic peptides derived from hypervariable V3 loops of distinct HIV-l isolates was studied and related to amino acid variability. It is recognized that for a more complete assessment of the impact of amino acid variability on protective immune responses, it will be necessary to study in great detail the cross-reactivity among HIV-l isolates, differing in primary sequence, at the level of virus neutralization and cytotoxic responses involving ACC and ADCC. Because of the complexity of such immunological studies, they will have to be limited to a few selected and well characterized subtypes (clones) of HIV-l. In this respect, studies concerning the serological cross-reactivity between peptides from distinct HIV-l isolates differing in primary sequence of the V3 hypervariable loops are expected to help in predicting the cross-protective potential of antibodies raised against peptides of defined sequence. The most extensive studies attempting to relate the degree of serological relatedness with the extent of sequence homology were carried out with plant viruses (Van Regenmortel, 1975, 1986) using polyclonal rabbit antisera against whole viruses. Similar studies using monoclonal antibodies (Altschuh et al., 1985; Dore et al., 1987) revealed that epitopes mimicked by linear peptides can actually be antigenically discontinuous and sensitive to mutations occurring outside of the recognized peptide sequence. In this respect it is of interest that the correlation between serological cross-reactivity and amino acid divergence within residues (9-37) (Fig. 1) corresponded to an apparent linear regression with a correlation coefficient r = 0.667, i.e. lower than the correlation coefficient calculated when the divergence of the full length sequence (144) was considered. This suggests that amino acid replacements within the N- and C-terminal parts of the sequence affect serological cross-reactivity, although short peptides from both the N- and C-terminus were apparently only weakly or not at all recognized by the antipeptide antisera. The discovery of neutralization escape mutants having hypervariable V3 loop sequences identical to HIV-l isolates which can be neutralized (Nara and Goudsmit, 1990) indicates that amino acid replacements outside the hypervariable loop can dramatically affect the recognition by antibodies of epitopes within the hypervariable loop. Further studies will be required to identify precisely the nature and location of these amino acid replacements. The scatter of points shown in Fig. 5 indicates that the relationship between serological cross-reactivity and amino acid divergence is not a straightforward immunogens,

it is necessary

to understand

547

from HIV-l

one and that some amino acid replacements affect serological specificity more than do others. Serological reactivities lower than expected from the linear regression [Fig. 5; log (% immunological crossreactivity) = 1.57 - 0.052 x divergence score] can to a great extent be ascribed to amino acid replacements at residues 14, 16 and 17 (Fig. 1). Broadly protective immunogens against HIV-l infection will have to contain a series of components expressing distinct epitopes. Peptides from the V3 hypervariable loop of judiciously selected HIV-l isolates are expected to become essential but not exclusive components of such complex immunogens. Results of serological cross-reactivity studies presented here, as well as the finding that it is possible to elicit broadly specific immune responses by combining several (21) subtype specific peptides into a single immunogen are expected to augment efforts aimed at development of strategies for prevention of AIDS. Acknowledgements-This study was supported by grant CA433 15 from the National Institutes of Health. We thank Dr R. Fields, J. P. Salley and Neosystem, Strasbourg. France for peptide synthesis; Dr A. Hellman and Dr A. Fowler, S.R.A. Technologies, Inc., Alexandria, Virginia for help in virus neutralization assays; T. Huima for photography, C. Stiles for word processing; Drs C. E. Stevens, P. Rubinstein and P. Taylor for anti HIV- 1 positive human sera; and L. Morgan for drawings.

REFERENCES

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Fig. 5. Dependence of the immunological cross-reactivity between all possible pairs of peptides listed in Fig. 1 (except the most recently sequenced JH-3 sequence) on the divergence score of the paired amino acid sequences. The dependence approximates a linear regression (correlation coefficient r = 0.718). The immunological cross-reactivities and divergence scores were calculated as described in Materials and Methods.

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