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(The FASEB Journal. 2001;15:2112-2120.)
© 2001 FASEB

Dissection of the humoral immune response toward an immunodominant epitope of HIV: a model for the analysis of antibody diversity in HIV+ individuals

DAVID ENSHELL-SEIJFFERS, LARISA SMELYANSKI, NURITH VARDINON*, ISRAEL YUST* and JONATHAN M. GERSHONI1

Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel; and
* Department of Clinical Immunology, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel

1Correspondence: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: gershoni{at}post.tau.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Understanding the dynamics of the humoral immune response to HIV epitopes in the presence of genetic drift and antigenic variation of the virus may reveal critical elements of protective immunity against HIV. Analysis of antibody maturation and diversity is difficult to study at the molecular level in humans. We used a combinatorial phage display peptide library to elucidate antibody diversity in HIV-infected individuals to a single immunodominant epitope in gp41. A serum sample derived from an HIV+ individual was used to screen a phage display a 12 mer cysteine-constrained loop peptide library. In doing so, we isolated mimotope-presenting phages corresponding to the immunodominant gp41 epitope CSGKLIC (residues 603–609). The mimotopes and control phages expressing epitope variants were reacted with a panel of 30 HIV+ sera. The patients showed distinct and variable recognition patterns compared with one another. Subfractions of the polyclonal sera were affinity purified and analyzed for epitope specificities. These analyses illustrated that epitope variants can be used to decipher antibody diversity. Elucidation of the plasticity of the humoral response and its polyclonality toward discrete epitopes contributes to our understanding of the antibody maturation process in individuals infected with viruses such as HIV.—Enshell-Seijffers, D., Smelyanski, L., Vardinon, N., Yust, I., Gershoni, J. M. Dissection of the humoral immune response toward an immunodominant epitope of HIV: a model for the analysis of antibody diversity in HIV+ individuals.


Key Words: AIDS • phage display • humoral response • peptides


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WHAT CONSTITUTES EFFECTIVE immunity against HIV-1 continues to be a central question for those interested in developing AIDS immunotherapeutics and preventives (1) . Cellular immunity is essential and considered the main arm of the immune system for the clearance of intracellular pathogens such as viruses (2 , 3) . However, it is becoming clear that antibodies play a critical role in the defense against HIV-1 (4 , 5) .

Measurement and characterization of serum antibodies can be highly prognostic. HIV-infected individuals with low neutralizing antiviral titers progress faster to disease than those that have acquired high levels of neutralizing anti-HIV antibodies in their blood (6) . Long-term nonprogressor HIV+ individuals characteristically produce broadly cross-reactive neutralizing antibodies (7) . Maternal transmission of viral infection from HIV+ pregnant mothers to their babies is inversely correlated with anti-HIV antibody titers of the mothers (8) . Direct evidence of antibody protection against HIV/SIV/SHIV infections can be generated when selected polyclonal immunoglobulin Gs (IgGs) or purified neutralizing monoclonal antibodies are used for passive protection in experiments with monkeys (9 , 10) and chimpanzees (11 12 13 14 15) .

In view of the above, much effort has been directed toward the identification of neutralizing viral B cell epitopes. Sera derived from HIV-infected individuals and murine and human anti-HIV monoclonal antibodies (mAbs) have been central to this (16 17 18 19 20 21 22) . Identification of epitopes recognized by HIV-infected individuals is often accomplished by screening their sera with synthetic peptides corresponding to HIV antigens for the presence of specific activity against these peptides. This, in turn, enables the affinity purification of the corresponding antibodies and determination of their neutralization activity (18) . However, mapping the epitopes of the murine and human mAbs first requires determining the target antigen and then testing the binding of the mAbs to antigen fragments, peptides, or mutants. An alternative method for mapping or identifying epitopes has been to use the mAbs of interest or specific sera to screen combinatorial phage display peptide libraries (23 24 25 26 27 28 29) .

HIV-1 has developed several strategies to evade immune surveillance such as down-regulation of MHC class I and cell surface CD4 (for a review, see refs 30 , 31 ). Moreover, HIV-1 constantly undergoes extensive genetic variation (32) . Concomitantly, a dynamic process of antibody maturation occurs (4) . This is mediated by somatic mutation and repertoire shift, which together modify the B cell response of the host so as to become focused on discrete epitopes of the virus (33) . Obviously, the latter can be counterproductive to the effort of clearing hypervariable viruses leading ultimately to escape mutants (34 35 36) , rendering the search for useful B cell epitopes difficult. Therefore, it is important to elucidate the dynamics of the humoral response toward neutralizing epitopes.

Once a particular epitope has been identified, numerous questions can be asked. How prevalent is the humoral response toward the epitope? Do all HIV-infected individuals see the epitope the same way? Do people generate a variety of different antibodies to the same epitope? Can different responses have different capacities for viral neutralization? During the course of the maturation of the humoral response, do novel variations in antibodies evolve with unique binding or neutralizing characteristics?

An interesting case in point is the humoral response to the major immunodominant epitope of HIV gp41, the constrained cysteine loop CSGKLIC (residues 603–609) (16 17 18 , 37 , 38) . This epitope is recognized by 90–100% of the HIV-infected individuals (16 , 17) . Numerous human mAbs have been isolated for this epitope and its vicinity (39 40 41 42 43 44) . Although the physiological significance of this particular epitope has been shown in some studies to be relevant to disease progression (18) , there is considerable debate as to whether mounting antibody responses to this epitope is beneficial or not. Thus, for example, Bugge et al. and Banapour et al. claim that human mAbs 41–7 (42) and 1B8.env (40) both bind this epitope, but are not neutralizing at all and "should be avoided in future vaccine candidates" (42) . Eaton et al. go as far as to claim that the mAb to this same region, 2F11, "appears to be the first anti-gp41 antibody that enhances infectivity of an HIV-1 strain" (44) . Yet Cotropia et al., who have reviewed this epitope in detail (43) , report that their human mAb "clone 3 and the conserved immunogenic epitope of gp41 could be useful in passive and active immunotherapy."

Thus, we are confronted with the fact that although most HIV-infected individuals see this epitope, they respond to it in very different ways.

We have focused on this immunodominant gp41 epitope as a model for studying antibody diversity toward a single epitope using a combinatorial phage display peptide library. In doing so, we evaluate the degree of antibody diversity and cross-reactivity directed toward the epitope in a panel of heterogeneous HIV-1+ individuals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Library construction
The ftac88 vector was used to construct a phage display library in which peptides are expressed as NH2-terminal fusion proteins of the PVIII coat protein of the filamentous phage, fd. The epitope library was built by introducing inserts that encode for random 12 mer peptides flanked between two constant cysteine residues between the vector’s two SfiI sites. The resulting complexity of the library was 9 x 108 recombinants.

Affinity selection of phages
Human polyclonal sera were heat-inactivated (56°C, 30 min) and diluted 1:100 in Tris-buffered saline (TBS) containing 0.25% (w/v) gelatin (TBSG). Protein G (Sigma Chemical Co., St. Louis, MO) was used to coat the bottom of a 35 mm petri dish overnight (70 µg/ml in 0.7 ml TBS, 4°C). After discarding the excess solution, the dish was blocked with TBSG for 2 h at room temperature, washed rapidly five times with TBS, incubated with the diluted serum (total volume 0.7 ml), and rocked gently at room temperature for 4 h. After washing with TBS, biopanning was accomplished by adding 1011 transducing units (TU) of phages from the library to the dish in 0.7 ml TBSG and incubated at 4°C overnight. Unbound phages were removed and the dish was washed extensively 10 times with TBS. Bound phages were eluted with 400 µl of elution buffer (0.1M HCl adjusted to pH 2.2 with glycine, 1 mg/ml BSA) for 10 min at room temperature with gentle agitation. The eluate was transferred into a 1.5 ml Microfuge tube and neutralized with 75 µl of neutralizing buffer (1M Tris-HCl pH9.1).

Eluted phages were amplified in DH5{propto}F' Escherichia coli and used for a second round of biopanning as described above.

Immunoscreening
DH5{propto}F' bacteria were infected with the affinity-selected phages (see above), plated on Luria-Bertani plates containing 20 µg/ml tetracycline, and grown at 37°C overnight. Single colonies were used to inoculate 200 µl Terrific Broth in U-bottom 96-well plates. After overnight culture, the plates were centrifuged at 3000rpm for 30 min at room temperature; 125 µl of the supernatant from each well was transferred to a flat-bottom, 96-well plate already containing 50 µl/well of polyethylene glycol (PEG)/NaCl solution (33% PEG, 3.3M NaCl). The flat-bottom plates were incubated at 4°C for 2 h and centrifuged. The precipitated phages were resuspended in total of 100 µl TBS and applied via a vacuum manifold to nitrocellulose filters. After blocking (5% evaporated spray dried skim milk 1.5% fat in TBS) for 1 h, the membranes were washed briefly with TBS and incubated overnight with serum diluted 1:10,000 in TBS/5% milk at 4°C with gentle rocking. After washing, the membranes were incubated with goat-anti human Fc IgG/HRP conjugate diluted 1:5000 in TBS/5% milk for 1 h at room temperature. The positive signals were detected by ECL (Amersham International plc, Buckinghamshire, England) immunodetection.

Generation of control phages #4, 7, and 8
The three control phages (#4, 7, and 8) were constructed by annealing two appropriate complementary oligonucleotides in order to create inserts containing 3' over hang ends compatible with SfiI sites of the ftac88 vector. Purified SfiI cut vector was used to ligate the inserts. The oligonucleotides used were as follows:

phage #4: 5'-TGGCTCTTTAGGTAAGCTTGTATCTGCCGCTG-3' 5'-CGGCAGATACAAGCTTACCTAAAGAGCCACGT-3';

phage #7: 5'-TGGCTGTTCAGGTAAGCTTATATGTGCCGCTG-3' 5'-CGGCACATATAAGCTTACCTGAACAGCCACGT-3';

phage #8: 5'-TGGCTCTTCAGGTAAGCTTATATCTGCCGCTG-3' 5'-CGGCAGATATAAGCTTACCTGAAGAGCCACGT-3'.

Quantitative ELISA
Microtiter plates were coated with rabbit anti M13 serum diluted 1:2000 in TBS for 17 h at 4°C. The plates were blocked with 5% milk in TBS for 2 h at room temperature and washed briefly with TBS. A total of 50 µl TBS containing PEG-precipitated phage was applied to the plates and incubated for 2 h at room temperature. After extensive washing with TBS, the plates were incubated with serum diluted 1:1000 for 24 h at 4°C and washed extensively with TBS. The plates were incubated with goat-anti human IgG/HRP conjugate diluted 1:5000 in TBS/5% milk for 1.5 h at room temperature. After washing, the plates were supplemented with O-phenylenediamine and incubated for 10 min at room temperature. The reaction was terminated with 4M HCl. OD490 was measured using an ELISA reader.

Analysis of phage affinity-purified antibody
A total amount of selected phages (5x1010) were applied onto nitrocellulose membrane filters using a vacuum manifold. After blocking for 1 h with 5% milk in TBS, the filters were incubated with sera diluted 1:10,000 (in the case of patient #I3287) or 1:20,000 (in the case of patient #I0998) in TBS/5% milk for overnight at 4°C. The filters were washed extensively with TBS; the captured antibodies were eluted with glycine-HCl buffer (pH 2.2) and neutralized with 1M Tris-HCl pH9.1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Screening patient’s polyclonal serum
Patient #I9809 is a female HIV+ individual who was infected by her husband heterosexually in 1988. Her viremia has fluctuated around 5000–7000 copies/ml and her CD4 count remains above 600. She is currently healthy and has not undergone any AIDS-related medical treatment. Her polyclonal serum was used to screen a phage display random peptide library expressing random 12 mer peptides flanked by constant cysteine residues expressed at the NH2 terminus of the PVIII major coat phage protein.

A single round of biopanning was performed and a total of 400 phages were picked and screened as dot blots against the serum. Only one positive phage was detected. Therefore, a second round of amplification and biopanning was performed and 600 phages were picked and screened. Of these, 24 phages proved positive and their corresponding inserts were sequenced. As can be seen in Fig. 1A , a collection of six different inserts could be defined. Three of the phages selected turned out to be cysteine-constrained pentamers (phages #1–3). In the course of constructing phage display libraries synthetic oligonucleotides were prepared. Although the majority of the oligonucleotides are of the specified desired length, shorter oligonucleotides are generated. In this way, the library contains a spectrum of constrained loops of different sizes. Therefore, the three 5 mer constrained loops indicate a strong selective advantage for this relatively rare configuration in our library. They are highly homologous among themselves and show linear homology of the LGKXXC motif in phage #9. The additional two phages (#5 and #6) seem to have no common motif.



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  		Figure 1. Sequences and homologies of isolated phages. Six phages were isolated by screening the library with the polyclonal serum of patient # I9809 (phages # 1–3, 5, 6, and 9). A) Sequences are given and ranked according to the frequency at which they were found, then according to the binding to the original serum. B) HIV consensus sequences for the gp41 loop residues 603–609. p197 is the phage isolated by Scala et al (31) . C) Sequences for the three control phages constructed for this study. Note that the common glycine-lysine central residues of the loop are highlighted for convenience.

The sequences of the isolated inserts were used to search for homologous stretches in HIV. Although no such homologies could be identified for phages #5 and #6, a good correspondence was discovered for the pentameric loops in the gp41 sequence (residues 603–609). The ability to affinity isolate peptide-displayed phages homologous to this gp41 sequence was not surprising, as this sequence is known to be an immunodominant epitope. As is shown in Fig. 1B , the consensus motif among HIV-1 clades is extremely well conserved for this epitope. Also, Scala et al. (45) previously isolated one mimotope for this region (Fig. 1B , phage p197).

Recognition of the gp41 epitope in HIV-infected individuals
A panel of 30 sera of HIV-infected individuals was screened against the isolated phage-displayed mimotopes obtained above. As controls, three additional phage-displayed peptides were constructed (Fig. 1C ). Control phage #4 represents the insert of phage #3 in which the flanking cysteine residues are exchanged for serines, thus testing the necessity for the looped configuration. Phages #7 and #8 represent the clade B consensus sequence (CSGKLIC) with and without the flanking cysteine residues. A negative control phage, ftac88 phage, containing only wild-type PVIII was included in the analyses.

Dot blot analyses of the 10 phages described above were performed using nitrocellulose strips containing the phages and reacted with the 30 HIV+ serum samples and 9 negative control samples of HIV- individuals. None of the phages gave positive results with the HIV- control sera (data not shown). Figure 2 shows the binding capabilities of eight representative HIV+ sera. The binding of the original serum (patient #I9809) shows that all six library selected phages are recognized with different degrees. The control phages #4 and #7 are bound, showing that this patient can bind the bona fide gp41 loop and can tolerate the linear configuration of phage #3. However, the linear representation of the genuine gp41 epitope (phage #8) is not recognized. By comparing the serum responses of different patients toward the gp41 epitope, its mimetics and linear vs. looped configurations, one can identify a diversity in the ability to recognize this epitope. None of the other HIV+ individuals recognize phages #5 and #6, showing the uniqueness of these peptides for patient #I9809. It is not clear whether or not these peptides represent HIV-related epitopes. The reaction of #I9809-derived serum toward phage #9 was weak and unique as well.



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  		Figure 2. Dot blot analysis of representative sera reacted with the panel of 10 phages. Nitrocellulose strips containing dots of the 10 phages described in the text were reacted with sera of 8 patients as indicated in Fig. 1 . Phage #10 is a negative control, the ftac88 phage that contains only wild-type PVIII protein. Serum dilutions were 1:40,000 (#I3286, #I0998, #I2372) and 1:80,000 (#I9809, #I0840, #I3287, #I3295, #I0842); signals were produced via ECL.

All of the HIV+ patients (except one; see Fig. 3 ) recognized phage #7, i.e., the genuine gp41 epitope. As expected, the same is true for phage #3, which is most similar to the gp41 consensus motif. Phages #1 and #2 are recognized variably: some patients can bind both variants (#I3286) or only phage #2 (#I0998). Note, for example, patient #I0842, who virtually does not recognize these two phages. The ability to bind the linear vs. looped configuration is also variable (compare #I0998 with #I2372). This illustrates that whereas many HIV+ individuals recognized the looped configuration of the gp41 epitope only, some can bind both the linear and the loop conformations.



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  		Figure 3. Quantitative ELISA of 30 HIV+ sera. The 10 phages used in Fig. 2 were applied to ELISA wells and reacted with diluted (1:1000) polyclonal sera of 30 patients as described in Materials and Methods. The signals are expressed as the number fold increase binding for the phages #1–9 over the binding to the negative control ftac88 phage #10. The results are the average of 4 independent experiments. The average signals are shown as: {square} < 3; 3 <= (cross-hatched) < 5; 5 <= (stippled) < 10; 10 <= (diagonal stripes) < 20; 20 <= (waffle pattern) < 50; {blacksquare} >= 50. f represents the frequency in percent each phage is recognized.

A calibrated ELISA assay was used to compare the variations in responses quantitatively. The titer of each phage was carefully determined and equal amounts of each phage were applied to the wells precoated with rabbit anti-M13 polyclonal serum. The value for serum binding to a particular phage was measured and the entire experiment was repeated four times. The signals obtained were expressed as the number fold binding over the binding to the negative control ftac88 phage #10. Figure 3 shows the binding of the 30 HIV+ sera toward the 10 phages.

All but one patient (#I3285) recognized the gp41 epitope. However, this epitope is recognized by different HIV-infected individuals in distinct ways: 40% bind all three variants of the epitope (phages #1–3), 47% bind two variants (phages #2–3), and 10% bind only one variant (phage #3). Moreover, 27% bind phage #4 (i.e., the linear configuration of phage #3) and 27% recognize phage #8 (i.e., the linear configuration of phage #7). The ability to bind to linear vs. looped conformers does not necessarily correlate cross-reactivity with sequence variants. Thus, for example, whereas #I0998 binds phage #8 and #4 exceptionally well, no recognition of phage #1 is detected. The same pattern of recognition is seen for other patients (e.g., #I0839 or #I3289) but with less intensity. However, patients #I02 and #I3290 bind all the gp41-related phages, looped and linear. Furthermore, the ability to bind to the linear conformer of phage #4 does not necessarily mean that the patient’s serum has the ability to recognize the linear conformer of phage #8, and vice versa. For example, #I3289 binds phage #8 but not phage #4, whereas #I3301 binds phage #4 but not phage #8.

Clinical status of the infected patients and phage binding
The patients were divided into three groups according to their ability to bind phages #1–3: Group A bound phage #3 alone (e.g., #I0842); Group B bound phages #2 and #3 (e.g., #I2368); and Group C bound all three phages (e.g., #I3286). The history of each patient and current clinical status were evaluated in an attempt to discover any correlate with gp41 epitope binding. No such correlate could be identified. For each group of patients defined above, some are healthy, some were never treated, and others were failing treatment with severe presentation of AIDS disease. No correlate could be discovered for the ability to bind (or not) the linear configuration of the epitope with clinical status.

Longitudinal analysis of selected patients
To evaluate whether the ability to recognize the gp41 epitope may change longitudinally, serum samples taken at different times from patients #I9809 and #I0842, #I2378, #I3285, #I0036 were tested. The last four patients were selected because they all showed poor responses toward phages #1 and #2 and low activity (or none, patient #I3285) for phages #3 and #7. For all five patients tested, recent serum samples (within the last 6 months) were included in these analyses and compared with the oldest available samples dating from 1997–1998. The greatest degree of change over time could be seen in patient #I9809, as illustrated in Fig. 4 . The differences observed are at best marginal. The other patients showed markedly less variation in their responses over time. It is important to point out that these latter four patients had all been on combination therapy throughout the period in which the blood samples were taken. Their viremia values have remained extremely low (below 100 or undetectable) and their CD4 counts have been in the range of 300–700.



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  		Figure 4. ELISA analyses of patient I9809 serum samples over time. Serum samples of patient I9809 from March 1998, April 1999, and May 2001 were diluted 1:1000 and tested in a quantitative ELISA test as described in Materials and Methods. The experiment was conducted twice and in duplicate; values given are the average of the four measurements. Binding to selected phages is shown.

Polyclonality of the immune response
The question of whether the ability to bind epitope variations and configurations was due to cross-reactivity of single antibodies or to the existence of polyclonality, i.e., different B cell clones recognizing different epitope variants, was addressed. For this, the sera of patients exhibiting different patterns of gp41 epitope recognition were analyzed. For example, see Fig. 5 A illustrating the analysis of patient #I3287, who recognized all four of the cysteine looped configurations of the gp41 epitope yet did not bind the linear variants (phages #4 and 8). Phages #1–3 were used individually to affinity purify phage-selected antibodies from the polyclonal serum of this patient. The antibodies derived from each separate phage were then used to screen all 10 phages in the ELISA assay described previously. Irrespective of the origin of the antibodies tested (Fig. 5A ), the pattern of binding to the different phages was the same. Thus, for example, phage #1 continued to be less recognized by antibodies regardless of whether they were affinity derived from phage #1 itself or the others. Similarly, phage #7 was recognized best irrespective of the source of the antibody being tested. Therefore, one concludes that binding of the different epitope variants is the result of antibody cross-reactivity and the flexibility of a given antibody to conform and tolerate antigenic variation.



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  		Figure 5. Analysis of purified phage-specific antibodies. A) Phages #1–3 were used separately to affinity purify phage selected antibodies from the serum of patient #I3287. The antibodies derived from each phage were then used to react with all 10 phages in the ELISA assay used in Fig. 3 . Binding to phages 5, 6, and 9 (not shown) was similar to that of phage 10. B) Phages #7 and #8 were used separately to affinity purify antibodies from the serum of patient #I0998 and the latter were used in ELISA assay as in panel A.

Another example of such an analysis of patient polyclonal serum is shown for patient #I0998, who recognized both the linear and the looped configurations (Fig. 5B ). Here antibodies were affinity purified from phages #7 and #8. In this manner, one could evaluate whether polyclonality is required for the ability to bind the linear conformation of the epitope as opposed to the looped structure. The antibodies eluted off phage #7 bound this phage the best and phages #2 and #3 quite well, as expected. Moreover, the pattern of recognition is similar to that obtained for the eluted antibodies of patient #I3287 with phages #1–3 (see foregoing text and compare panels A and B in Fig. 5 ). The binding of phages #4 and #8 was less than threefold more than the binding of the negative control phage #10, and thus considered insignificant. Therefore, the antibodies eluted with phage #7 recognize the gp41 epitope looped configuration only. However, the results for the phage #8 affinity-purified antibody showed that the ability to bind this phage is considerable. Moreover, the phage #8-derived antibodies are able to cross-react markedly with phages #2, #3, and #7, indicating that these antibodies are capable of binding the linear as well as the looped epitope conformations. Furthermore, the recognition pattern is distinct from that observed for the phage #7-derived antibodies. The phage #8 affinity-purified antibodies bind to phages #2, #3, and #7 at approximately the same intensity vs. the drop in intensity characterizing the binding of phage #7-derived antibodies to these phages. However, the phage #8-derived antibodies are not able to cross-react with phage #4. This is surprising, as one might have expected that the antibodies that recognize phages #8 and #3 should be able to recognize phage #4 as well. This is especially true, as anti-phage #4 activity is clearly present in the patient’s polyclonal serum (see Figs. 2 and 3 ). Thus, one can conclude that at least three clonal types of anti-gp41 antibodies must exist for patient I0998: antibodies that bind the looped epitope exclusively, those that can bind the linear form of phage #8, and those that bind phage #4. One also can illustrate that various levels of cross-reactivity can exist for each class of antibody.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The phage display analysis of patient #I9809’s serum generated six peptides, three of which defined the gp41 immunodominant epitope CSGKLIC (residues 603–609). Scala et al. (45) previously demonstrated that polyclonal sera of long-term nonprogressors can be used to screen combinatorial phage display peptide libraries and they isolated a phage corresponding to the same gp41 loop. Moreover, they clearly showed that such phages can in turn be used to affinity purify the neutralizing IgGs from polyclonal sera. They also demonstrated that the phages could be used to elicit protective immunity.

In the current report we are not concerned with using the phage display technology for epitope mapping, i.e., cataloging what epitopes are recognized by a particular collection of polyclonal sera. Rather, we addressed a different question, namely, can phage display analyses be used to evaluate the degree of plasticity and polyclonality of the response mounted by the panel of 30 HIV-infected individuals toward a defined immunodominant HIV epitope? From previous studies of human mAbs toward the same epitope, one realizes that simply binding to the epitope might not necessarily render virus neutralization. The human monoclonal antibodies 1B8.env (40) and clone 3 (43) both bind the same gp41 epitope. However, clone 3 is capable of neutralizing several HIV isolates and 1B8.env is not. This illustrates that a single epitope can elicit antibodies with different biological activities. We propose that this type of diversity in the ability to recognize epitope structural variants could play a role in counteracting viral genetic drift as well.

As Fig. 3 illustrates, focusing on a single phage can be misleading. The frequency of recognition of phage #1, for example, is 40% and could be interpreted to mean that a significant proportion of HIV-infected individuals do not ‘see’ this site (note that the recognition frequency of phage p197 in Scala’s report is 59%). The issue of how frequently a particular epitope is actually recognized becomes more accurately discernible by using a collection of epitope mimetics and defined control peptides. The collection of epitope variants used in this study illustrates clearly, as has been reported previously (16 , 17) , that 97% of the patients reacted toward the gp41 loop.

The three phages (phages #1–3) represent different aspects of the same CSGKLIC epitope. One could expect that if a polyclonal serum recognizes one phage, it should bind the other two. As can be seen in Fig. 3 , the recognition capabilities of different patients are quite variable. This suggests that a single epitope can elicit antibodies with quite different binding characteristics. This also illustrates that a collection of epitope variants can enable one to assess the degree of flexibility of the response to structural deviations from what might have been the ‘original sin’ or the degree of genetic drift within the quasi-species.

The question of change of the immune response over time was also addressed. Patient #I9809 continues to be healthy and has not been treated with any antiretrovirals. Her viremia has been as low as 600 (December 1998) and as high as 7000 (December 1996) and is generally on average in the range of 4000–6000 over the last 5 years. Only in her case was there any detectable progression in the ability to bind the gp41 epitope variants. These variations are minimal, yet may show some development of response as is illustrated in Fig. 5 . More important, however, is the fact that the four patients that did not show any progression are all being treated with combination therapy. Their viremia has been barely detectable; thus, it may be that in the absence of high titer of virus, i.e., an immunogenic stimulus, the humoral immune response cannot progress. This might be particularly important when considering Structured Therapeutic Interruptions. To enable progressive improvement of the immune response, it might be necessary to allow the viremia during the interruptions to rise to a reasonable level so as to stimulate the immune system before initiating the next period of therapy.

The selective affinity isolation of phage-specific antibodies and measurement of their abilities to bind to the different phages enabled us to distinguish between the cross-reactivity of given antibodies for epitope variants as opposed to the existence of distinct polyclonal variations in the response. For example, one patient (#I0998) expresses at least three clonal types of antibodies that react with the gp41 epitope, each in a different manner. This polyclonality can be a result of either antibody maturation or activation of different independent B cell clones. Obviously, generating more expansive collections of epitope variants would increase the resolution of our assays accordingly and allow one to evaluate the fine specificity of a patient’s response and the overall profile of the general utility of a given epitope. One could also identify epitope variants that might be effective diagnostics/prognostics as well as potential subunit vaccine candidates.

Elaborate studies in these directions have been performed when studying the humoral response toward human hepatitis virus type C (HCV) (46 47 48 49) . Initially, Folgori et al. demonstrated the use of polyclonal sera in screening phage display libraries (28) . This was done in the model system comparing sera derived from individuals immunized and nonimmunized with the human hepatitis B virus envelope protein (HbsAg). Prezzi et al. applied the same strategy using sera from HCV-infected patients and uninfected subjects to identify peptides specifically reacting with patient’s sera (46) . Since then, numerous studies have been published in which detailed analyses of the response to HCV epitopes and the subsequent use of selected peptides as diagnostics and vaccines have been described (47 48 49) . The study by Puntoriero et al. in which the hypervariable region 1 (HVR1) of the E2 protein of HCV is analyzed is particularly noteworthy (49) . Here, the investigators first evaluated a collection of more than 200 HVR1 sequences to determine a consensus profile of the epitope. Then a novel library of phage-displayed peptides representing a large collection of epitope surrogates was constructed and used to identify particular epitope variants with desired characteristics. Urbanelli et al. describe a systematic methodology to generate epitope collections based on a defined mimotope (48) . These investigators describe a process of ‘reversed’ affinity maturation of the ligands for the paratopes of particular antibodies.

Indeed, the issue of the maturation of the immune response toward such a hypervariable virus as HIV is important to understand. Such maturation is characterized by an increased affinity and expanded cross-reactivity (4) . For example, Cole et al. have demonstrated that the evolution of protective immunity in monkeys inoculated with attenuated strains of SIV is associated with a complex and lengthy maturation of the humoral response over a prolonged period of 6 to 8 months postinoculation. This maturation is reflected in progressive increase in antibody titer and avidity (50) . The extent of polyclonality of the response toward a given target must also be considered. These researchers have illustrated that the maturation process of the antibody response is associated with changes in antibody conformational dependence. Whereas envelope-specific humoral response is always predominantly directed to conformational epitopes, the evolution of protective immunity is accompanied by an increase in antibodies that specifically bind linear determinants, suggesting a broadening of the humoral immune response to include recognition of additional epitopes and thus expanding polyclonality.

Of particular relevance are the works of Zinkernagel and colleagues in which the response toward vesicular stomatitis virus (VSV) has been studied in mice (51 52 53) . In contrast to the maturation of the humoral response to hapten ligands such as 2-phenyl-5-oxazolone, in which antibody maturation coincides with increased affinity (54) , this process toward viral protein antigens tends to be less dramatic. The primary response toward VSV can be of relatively high affinity and the subsequent somatic mutations might not necessarily contribute to acquisition of higher affinity antibodies (51) . We suggest that in the case of antibody maturation toward a viral epitope, the result of somatic mutations may be to increase the capability to cope with pathogen genetic drift rather than to enhance binding affinity. Such a mechanism would lead to the antibody diversity observed. Another mechanism for increased cross-reactivity toward viral variants could include the anti-idiotypic network (55 , 56) . The mAb M77 (57) binds specifically to the HIV-1IIIB gp120 V3 loop and neutralizes it. The Ab2{alpha} anti-idiotypic mAb, GV12, recognizes M77 and by so doing enables M77 to bind and neutralize a variant of HIV-1, which is not recognized by M77 alone. Clearly, the development of effective protective humoral responses toward viruses can be very complex. However, elucidation of these processes should contribute to the definition of what constitutes neutralizing immunity and how to achieve it.

The study of the development of antibody diversity toward viral epitopes is accomplished in animal models where splenocytes can be isolated and fused systematically after defined immunization regimens, and the molecular structures of VL and VH peptides can then be determined. In HIV infection, obviously one cannot conduct such systematic analyses to reveal the dynamics of the maturation and diversification of the humoral response. We describe here a model for the study of antibody diversity developed in HIV patients. Selected polyclonal sera are used to screen combinatorial phage-displayed peptide libraries. Panels of epitope variants and constructions of their corresponding consensus peptides are then screened with collections of patients’ sera. By expanding the repertoire of epitope ‘mini-libraries’ and correlating the use of given targets by patients with different disease statuses, one might be able to recognize the elements that constitute the protective immunity toward HIV.


   ACKNOWLEDGMENTS
 
This study was supported by the Israeli Ministry of Science, The Israel Science Foundation, and the DA’AT Consortium.

Received for publication January 2, 2001. Revision received June 19, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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