Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies

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1 Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies Alexandra Trkola 1, Herbert Kuster 1, Peter Rusert 1, Beda Joos 1, Marek Fischer 1, Christine Leemann 1, Amapola Manrique 1, Michael Huber 1, Manuela Rehr 2, Annette Oxenius 2, Rainer Weber 1, Gabriela Stiegler 3, Brigitta Vcelar 3, Hermann Katinger 3, Leonardo Aceto 1 & Huldrych F Günthard 1 To determine the protective potential of the humoral immune response against HIV-1 in vivo we evaluated the potency of three neutralizing antibodies (2G12, 2F5 and 4E10) in suppressing viral rebound in six acutely and eight chronically HIV-1 infected individuals undergoing interruption of antiretroviral treatment (ART). Only two of eight chronically infected individuals showed evidence of a delay in viral rebound during the passive immunization. Rebound in antibody-treated acutely infected individuals upon cessation of ART was substantially later than in a control group of 12 individuals with acute infection. Escape mutant analysis showed that the activity of 2G12 was crucial for the in vivo effect of the neutralizing antibody cocktail. By providing further direct evidence of the potency, breadth and titers of neutralizing antibodies that are required for in vivo activity, these data underline both the potential and the limits of humoral immunity in controlling HIV-1 infection. Humoral immunity in concert with cellular immune responses is thought to be required for natural and vaccine-induced control of HIV-1 infection in humans 1,2. Although ample data exist showing that neutralizing antibodies can protect against HIV-1 infection in vitro 3,4 and in animal models in vivo 5 14, proof of their activity in infected humans remains circumstantial and is best evidenced by the rapid selection of neutralizing antibody escape variants Efforts to generate vaccines based on humoral immunity are underway but so far have not elicited broad and potent neutralizing antibody responses 32. Hence, the potential of the humoral immune response in protecting against infection and controlling established human HIV-1 infection remains indeterminate. To formally define the activity of neutralizing antibodies in established infection we initiated a nonrandomized and unblinded proof-of- principle study in which three neutralizing monoclonal antibodies to HIV-1 one antibody to gp120, 2G12 (refs ), and two antibodies to gp41, 2F5 (refs ) and 4E10 (refs. 34,39) were passively administered over 11 weeks to HIV-1 infected patients who carried viral isolates with high sensitivity to the three antibodies. Subjects were on ART before antibody administration. ART was interrupted after the first antibody infusion and the capacity of the antibodies to suppress or delay viral rebound was evaluated. The antibodies 2G12, 2F5 and 4E10 have been shown to potently inhibit HIV-1 in vitro and in animal studies in vivo 5 7 and have recently undergone phase 1 clinical testing By passively transferring these antibodies before treatment interruption, we mimicked conditions after a successful therapeutic vaccination and were able to examine whether neutralizing antibody responses in principle have the capacity to suppress HIV-1 replication in vivo. RESULTS Passive administration of neutralizing antibodies We recruited 14 subjects from 58 acutely and chronically HIV-1 infected and ART-treated individuals whose autologous virus isolates had been tested in vitro for sensitivity to the neutralizing monoclonal antibodies 2G12, 2F5 and 4E10 (Table 1 and Supplementary Table 1 online) 41. Starting 1 d before ART was stopped, the selected eight chronically and six acutely infected individuals received 13 passive immunizations with the three monoclonal antibodies over an 11-week period (Fig. 1). Antibody infusions were well tolerated in most subjects; mild side effects were reported only occasionally (Supplementary Note online). After completion of the passive immunization phase, subjects were further followed up to week 24 and closely monitored immunologically and virologically. We measured plasma levels of the passively administered monoclonal antibodies throughout the trial. With the exception of subject NAB09, plasma levels for 2G12 were considerably higher than those for 2F5 and 4E10 (Fig. 1 and Supplementary Table 2 online). As described previously 20 22, 2G12 had a higher elimination half-life and consequently accumulated over time and was detectable in 11 of the 14 subjects at levels above 20 µg/ml at week 24 (Fig. 1 and Supplementary 1 Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Switzerland, Ramistrasse 100, 8091 Zurich, Switzerland. 2 Institute for Microbiology, ETH Hönggerberg, HCI 4 Wolfgang-Pauli-Strasse 8093 Zurich, Switzerland. 3 Polymun Scientific, Nussdorferlände 11, 1190 Vienna, Austria. Correspondence should be addressed to A.T. (alexandra.trkola@usz.ch) or H.F.G. (huldrych.guenthard@usz.ch). Published online 8 May 2005; doi: /nm1244 NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1

2 Table 1 Subject characteristics summary data Before last ART Baseline of passive immunization trial Subject group Age (years) Gender Mode of transmission a RNA b (c/ml) Months of VL <50 copies/ml CD4 cells/mm 3 Body weight HIV subtype male transmission 1 transmission 2 transmission 3 subtype B Chronic passive immunization Acute passive immunization Acute control group 46.5 (36 57) 29 (21 47) 39 (27 59) 62.5% 62.5% 12.5% 25% 55,537 (1,387 1,069,200) 83% 67% 33% 0% 83,850 (13,494 2,599,519) 75% 50% 42% 0% 28,290 (3,174 1,805,000) Table 2 online). We confirmed that the in vitro plasma neutralizing activity in the subjects sera agreed with the level of activity predicted by the pharmacokinetic measurements of plasma concentrations of the three monoclonal antibodies (data not shown). Effect of neutralizing antibodies in chronic HIV infection For the eight participating subjects with chronic HIV-1 infection, viral rebounds in the absence of antibody infusion had been intensively studied during previous structured treatment-interruption trials 42,43. The timing of the viral rebounds during these previous interruptions was stable over prolonged periods of time (Supplementary Table 3 and Supplementary Fig. 1 online). This allowed us to compare viral rebounds in the absence or presence of passively administered antibodies in the same subject and to determine whether and to what extent neutralizing antibodies are capable of delaying or suppressing viral replication. In six subjects (NAB01, NAB02, NAB05, NAB06, NAB07 and NAB09), viral replication during passive immunization closely mirrored previous interruptions (Fig. 1). No influence of the antibody treatment was evident as judged by the timing of the rebound during antibody therapy. The most pronounced effect of the neutralizing antibody treatment was evident in subject NAB03, whose viremia rebounded in previous treatment interruptions after 2 4 weeks. In contrast, administration of neutralizing antibodies completely inhibited viral rebound in this subject throughout the entire period of passive immunization. Virus became first detectable at week 18, at a time when antibodies 2F5 and 4E10 had already been washed out (0.6 and 1.3 µg/ml, respectively) and levels of 2G12 had dropped to 179 µg/ml. Viral loads then increased continuously and reached the previous set point by week 24. Similarly, subject NAB04 showed a considerably delayed viral rebound. In previous treatment interruptions, virus reemerged in this subject within 1 week, whereas during antibody therapy viral rebound virus became detectable only by week 6 (Supplementary Table 3 and Supplementary Fig. 1 online). Equally, peak viremia was delayed by 4 5 weeks, indicating a clear albeit transient effect of the neutralizing antibodies in controlling viremia. 4 (3 21) 531 ( ) 12 (6 14) 521 ( ) 16 (11 25) 847 (504 1,419) % ( ) 79 50% ( ) 72 (51 94) Statistical comparison acute passive immunization group and acute control group P value (Mann Whitney) n.s. (P = ) n.s. (P = ) P = P = 0.01 n.s. (P = ) P value (Fisher exact test) n.s. (P = 1) n.s. (P = 1) b n.s. (P = 1) c a Modes of transmission: 1, homosexual; 2, heterosexual; 3, intravenous drug abuse. b Frequency of transmission modes 1 and 2 in the individual groups tested with Fisher exact test. c Comparison of frequency of subtype B and non-b isolates. VL, viral load. 50% Effect of neutralizing antibodies in acute HIV infection Amongst the six subjects with acute HIV infection who participated in the passive immunization trial, the most rapid rebound occurred in subject NAB10, whose virus load rose above the threshold of 50 RNA copies/ml by week 5 (Fig. 1). In subject NAB11, viremia became detectable after 6 weeks and reached a peak value of 69,300 RNA copies/ml by week 9, which matched the pre-art viral load. In contrast, in subject NAB12, virus was also measurable by week 6 but levels stayed low (<100 RNA copies/ml) until week 9. Subsequently, viremia rose gradually and reached peak levels at week 12. Notably, this maximum level was 2 logs lower than the recorded pre-art viral load. The remaining three subjects (NAB08, NAB13 and NAB14) controlled viremia under passive immunization for prolonged periods of time. Viremia in subject NAB08 was first detectable at week 9 (6 RNA copies/ml) and then rebounded rapidly to reach peak values at weeks 11 and 14. Subject NAB13, who had a high pre-art viral load ( RNA copies/ml), controlled viremia almost for the entire passive immunization period. Viremia was detected at 7 and 5 RNA copies/ml at weeks 11 and 12, respectively, but rose to levels above 100 RNA copies/ml by week 13, when plasma levels of the passively administered monoclonal antibodies were already declining. Most noteworthy, subject NAB14 controlled viremia throughout the whole observation period. At a single time point (week 14) we detected a viral load of 11 copies/ml but afterward viral load remained undetectable. The evaluation of treatment success in the acute cohort is more complex because autologous subject data from previous rebounds were not available, as ART was interrupted for the first time during the passive immunization trial. Additionally, when evaluating viral loads before ART and after antibody treatment, it has to be considered that these subjects possibly had not reached a steady state before initiation of therapy and thus pre-art viral load levels may not adequately reflect their viral set point. To evaluate the efficacy of neutralizing antibodies in suppressing or delaying viral rebound in acutely infected individuals, we therefore compared the rebound in acute subjects undergoing passive immunization with a control group of 12 acutely infected individuals who interrupted ART without receiving passive immunization 2 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

3 Figure 1 Analysis of viral rebound upon cessation of ART in 14 passively immunized subjects. Individual profiles for the 8 chronically and 6 acutely infected individuals participating in the passive immunization trial are shown. Viral load is the log HIV RNA copies/ml in plasma. Red symbols depict longitudinal viral load measurements during the passive immunization trial. Black squares with red shading indicate the detection limit of the viral load measurement on a given day and are shown at time points where viral loads were not detectable. Viral loads measured during rebound of viremia in previous treatment interruptions in the absence of any treatment are depicted in blue, green and black (the three colors depict 1 3 profiles from independent ART interruption intervals 42,43 ). For chronically infected subjects the dashed gray lines depict the geometric means of the viral load before the last ART (Supplementary Table 1 online). For acutely infected subjects the dashed gray lines depict the last recorded viral load before initiation of therapy. Violet, blue and orange shaded areas indicate plasma concentrations of 2F5, 4E10 and 2G12, respectively. (Fig. 2 and Supplementary Table 1 online). Of note, subjects in the acute group receiving passive immunization and the control group were not matched for antibody sensitivity, nor were they randomized; thus confounding factors cannot be completely excluded. Notably, subjects in the acute intervention and control group showed no significant difference in pre- ART viral loads (Table 1 and Supplementary Fig. 2 online). The duration of successful ART, and consequently also CD4 levels, at the baseline of the treatment interruption were slightly higher in the control group as compared with the acute subjects in the passive immunization trial. But this discrepancy had no influence on our analysis, as we found no interdependency between length of ART and the timing of the rebound (Supplementary Fig. 2 online). As described previously 44, we noted that reemergence of viremia upon cessation of ART can be less rapid and at lower levels in subjects who received ART during early disease stages (Figs. 1 and 2a and data not shown). Nevertheless, rebound under passive immunization was substantially slower than in absence of antibody treatment. Viral rebound at levels above 10 RNA copies/ml in the control group ranged from 2 to 12.9 weeks (median, 3.75 weeks), whereas under antibody treatment time to rebound was significantly higher (range, 5 to >24 weeks; median, 8 weeks; (P = ) Fig. 2b). When we dichotomized the subject groups at week 5, which was the most rapid rebound above 50 viral RNA copies/ml observed amongst individuals in the intervention group, we found that 8 (67%) of the 12 control subjects rebounded at levels above 50 RNA copies/ml in less than 5 weeks, whereas none of the antibody-treated subjects rebounded as quickly (Fisher exact test, P = ). Of note, viral loads in the control group were measured in 2 4-week intervals, whereas viral loads in the passively immunized subjects were measured in 1- (week 0 12) to 2-week (week 12-24) intervals. Hence, our analysis in the control group is biased toward late detection of viremia and rebounds may have occurred in several instances earlier than recorded. Thus the differences in rebounds between the two groups are potentially larger than documented here. Viral escape Viral rebound despite the presence of neutralizing antibodies could indicate either that antibodies were ineffective in vivo, in which case antibody-sensitive viral strains would persist, or alternatively, that the virus escaped the antibody pressure. To investigate whether antibodies induced immune selection, we compared the inhibitory activity of the three monoclonal antibodies against virus isolates derived before the passive immunization trial with the activity against sequential isolates derived under antibody treatment (Fig. 3 and Table 2). We observed no changes in the sensitivity to 2F5 and 4E10 for any of the viruses derived from antibody-treated individuals nor did we detect relevant sequence changes in the core epitopes of these antibodies in viral genome sequences amplified directly from subjects plasma and from viral isolates (Fig. 3, Supplementary Table 4 and Supplementary Methods NATURE MEDICINE ADVANCE ONLINE PUBLICATION 3

4 a b online). In addition, no decrease in the activity of 2G12 was found in subjects NAB09 and NAB14, who carried isolates that were initially relatively insensitive to 2G12 (Fig. 3, Supplementary Tables 1 and 5 online). Conversely, in the remaining 12 subjects we observed a pronounced resistance of the rebounding virus to 2G12, which in several cases gradually increased over time. Although influence of the autologous immune response in escape selection cannot be completely excluded, these data are highly suggestive of an immune escape induced by 2G12. Of note, treatment interruption in the absence of 2G12 did not lead to frequent emergence of 2G12-resistant strains (Supplementary Table 5 online). Notably, 2G12-sensitive strains were detected with decreasing 2G12 levels in subjects NAB09 and NAB10. The fact that virus in subjects with comparatively early rebound (NAB01, NAB02, NAB05, NAB06, NAB07, NAB10, NAB11) as well as virus that emerged after prolonged suppression of viremia (NAB03, NAB04, NAB08, NAB12, NAB13) had escaped inhibition by 2G12 strongly suggests that this antibody was crucially involved in mediating viremia control. Analysis of treatment success To analyze which parameters influenced the individual response to the antibody treatment, we grouped subjects according to their treatment success, which was exclusively defined on the basis of a delay in viral rebound. Amongst chronically infected individuals we classified subjects NAB03 and NAB04 according to their delayed rebound compared to previous interruptions (Fig. 1) as responders, and NAB01, who showed a moderately decreased viremia but no delay of rebound, and the remaining subjects as nonresponders. In the acute group clearly classifiable responding subjects were NAB08, NAB12, NAB13 and NAB14, who all had a prolonged delay of viral rebound (>8 weeks below 100 HIV-1 RNA copies/ml plasma). We found no difference between the two groups in terms of pre-art viral loads, CD4 levels at baseline of the trial or gender (Fig. 4a,b and data not shown). Efficacy of the passive immunization is probably determined by two factors: the sensitivity of the given virus strains to inhibition by the antibody and the antibody concentration achieved in vivo. Although antibody dosage was not adjusted to each subject s body mass, plasma antibody levels were not significantly influenced by differential body weight Figure 2 Comparison of virus rebound in acutely infected subjects with and without passive immunization. (a) Control group of subjects with acute HIV infection undergoing treatment interruption: viral load profiles of 12 subjects who initiated ART during acute infection and subsequently underwent treatment interruption without receiving antibody treatment are shown. Viral load is the log HIV RNA copies/ml in plasma. The dotted line indicates the detection limit. (b) Time until rebound of viremia (first time viral load detectable at >10 RNA copies/ml, increase over day 0 value) was determined in the control group and in acutely infected subjects who received passive immunization. The fraction of subjects without rebound at a given time point was compared using Kaplan-Meier curves and log rank test. One data point (subject NAB14, week 24) was censored because rebound had not occurred in this patient. (data not shown). Overall, plasma levels of 2G12 were significantly higher than those of 2F5 or 4E10 (Dunn multiple comparison test, P < and P < 0.01, respectively). Of note, plasma concentrations of 2G12 and the sensitivity of the subjects pretrial isolates to inhibition by 2G12 were higher amongst responders, whereas no significant difference in these parameters was found for 2F5 and 4E10 (Fig. 4c,d). To estimate what range of antibody concentrations are required to suppress viral replication in vivo compared to in vitro we analyzed by which factor the plasma concentrations (PC) of the monoclonal antibodies exceeded the in vitro determined inhibitory doses (ID 90 ; Fig. 4e). No differences in this ratio were found for 2F5 and 4E10, whereas responders had significantly higher PC/ID 90 ratios for 2G12 (P = ). Most notably, 2G12 levels in plasma were an average of 394-fold higher than the in vitro ID 90 in responders compared to a factor of 36 in nonresponders, suggesting that 2G12-mediated control may be key to the sustained inhibition observed in responding subjects. The notable exception amongst responders was subject NAB14, who successfully suppressed viral rebound despite harboring a 2G12-resistant virus strain before the passive immunization. Control of viremia in this subject, if induced by the antibody treatment, must therefore have been through 2F5 and 4E10. In support of this, the plasma concentration of 4E10 exceeded in vitro inhibitory doses in this subject by a factor of 150, a value similar to that from subject NAB13, in whom the plasma concentration of 4E10 was 152-fold higher than in vitro inhibitory doses, the highest value amongst 4E10 PC/ID 90 ratios. DISCUSSION Our experimental approach in determining the role of the humoral immune response in HIV-1 infection was to evaluate the efficacy of passively transferred neutralizing antibodies in suppressing viral rebound in individuals undergoing interruption of ART. By enrolling subjects who carried viral isolates with high sensitivity to the administered antibodies, we had the opportunity to examine the activity of the humoral immune response under conditions that guarantee maximal potency of the antibodies. Two chronically and four acutely infected individuals showed evidence of a pronounced delay in viral rebound during antibody treatment, suggesting that neutralizing antibodies can, in principle, contain viremia in established HIV infection. Despite the fact that subjects harbored virus isolates that were initially sensitive to the monoclonal antibodies, virus rebounded in the remaining subjects without substantial impact of the antibody treatment. Overall, the impact of antibody treatment seemed to be more pronounced among acutely infected individuals, a fact that could be attributed to a more confined viral population in these individuals, less apt to give rise to rapid escape mutants. Although our study was neither randomized nor blinded, and thus more prone to confounding factors, our data strongly suggest that neutralizing antibodies can be effective in established HIV-1 infection. 4 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

5 Table 2 Evolution of 2G12 escape mutants Chronic Acute NAB01 NAB02 NAB03 NAB04 NAB05 NAB06 NAB07 NAB09 NAB08 NAB10 NAB11 NAB12 NAB13 NAB14 Week VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 VL 2G12 pre S S HS HS S S HS S/R HS HS HS HS HS HR 0 < 7 14 HS < 5 < < 7 < 8 < 8 < 7 < 5 42 < 6 < < 7 8 < 5 < < 7 < 8 < 7 < 5 < 6 < 7 < 6 < 11 1 < 5 42 < 8 < 10 < 11 < 7 36 < 6 < 10 < 6 < 4 < 6 < 8 < ,290 < 6 < 15 1,200 2, < 6 < 8 < 6 < 7 < 13 < ,500 < 6 < 10 29,600 16,800 1, < 6 < 13 < 10 < 17 < 9 < , ,000 HR < ,000 S/R 207,500 HR 17,500 R 39,100 < 6 HS 44 < 8 HS < 7 HS < 5 < ,500 55,900 < 6 < 15 47,300 85,900 54,700 72,000 < < 8 < 8 < 6 < ,400 73,700 < ,900 93, ,000 1,860 < < 6 < ,000 57,700 < ,100 66, ,000 2,710 < 10 13,900 8, < 7 < ,600 S/R 60,100 R < 8 HS 3,380 58,500 S/R 84,000 HR 151,000 3,260 S/R < 11 79,200 HR 18,400 HS 47 HS < 7 < , ,000 < 8 21,700 38, , ,000 3, ,800 69, < 6 < , ,000 < 7 25,400 63, ,000 92,500 5, ,600 48, < 6 < , ,000 < 6 8,300 37,500 94,900 53,600 5,390 21,300 54,700 21,000 1,590 7 < , ,000 < 10 9,570 74,300 91,700 S/R 70,000 R 4,050 6, ,000 24,000 12,000 R 5 < , ,000 < 7 116,00 71, ,000 30,000 18,700 8, ,000 19,800 10, < ,600 46,600 < 8 4,460 97, ,000 6,230 23, ,000 5,470 14, ,200 46,100 < 8 5, ,000 R 34,200 S/R 3,480 10,100 HR 102,000 S/R 15,800 5,290 HR 15 HS < ,500 69, ,440 67,900 54,500 15,400 3, ,500 9,260 1, < ,500 R 59, ,280 S/R 49,900 HR 35,100 R 9,330 HS 1,720 97,600 HS 13,200 2, R < ,500 69, ,430 55,300 41,500 8,440 1,950 51,000 7,720 2,190 < 8 < ,000 R 24 79,500 HR 3,110 S/R 2,030 84,900 HR 28,100 HR 9,850 1,680 HR 59,300 13,100 R 3, < 5 HR Sensitivity against inhibition by 2G12 of viral isolates derived from subjects CD4 cells at the indicated week are depicted alongside the viral loads (VL) in plasma at the same time points. Sensitivity to 2G12 was scored as follows: high sensitivity (HS), ID 90 < 10 µg/ml; sensitive (S), ID 90 < 25 µg/ml; semi-resistance (S/R), ID 90 > 25 µg/ml, ID 70 <25 µg/ml; resistance (R), ID 70 > 25 µg/ml, ID 50 < 25 µg/ml; high resistance (HR), ID 50 > 25 µg/ml. Monoclonal antibody 2G12 had a dominant effect on the study outcome: all 12 subjects with 2G12-sensitive virus strains developed escape mutants to this antibody, and failure to respond to antibody treatment, as well as loss of viremia control, was strongly associated with the emergence of 2G12-resistant viral strains. In several cases, these escape mutants emerged very rapidly and at high titers, indicating that 2G12 resistance can be attained easily and without apparent loss of fitness. These results support previous findings in animal studies in which antibody treatment was reported to be effective but also subject to rapid escape 11,14. In those individuals in whom 2G12-resistant virus rebounded without delay, we cannot, however, distinguish formally whether resistant strains were generated de novo or whether they represented preexisting, naturally occurring 2G12-resistant viruses that were readily selected during antibody treatment. Notably, no escape to the gp41 monoclonal antibodies 2F5 and 4E10 occurred in our study. Two hypotheses may explain this disparity in the generation of escape mutants. One hypothesis is that only the cocktail of the three neutralizing antibodies is capable of exerting selection pressure in vivo and as soon as activity of one of the antibodies is lost because of the emergence of resistant virus, the remaining two antibodies are too weak to control viremia and to induce selective pressure. In this scenario, 2G12 would be the weakest of the three antibodies, indicated by the rapid appearance of escape mutants. 2G12 escape has been previously shown to arise from the loss of one of the glycosylation sites within the carbohydrate epitope of the antibody In comparison, escape to 2F5 and 4E10 may be more difficult to achieve and/or result in loss of fitness in vivo. Another hypothesis is that because only 2G12 escape occurs, only this antibody may be active in vivo. Differences in the mode of action may exist in vivo that render 2F5 and 4E10 less effective. Alternatively, only 2G12 may have achieved the plasma concentration required for in vivo activity. In support of the latter hypothesis, we found that plasma levels of 2G12 were substantially higher than those of 2F5 and 4E10. Most importantly, in subjects who responded to antibody treatment, plasma levels of 2G12 exceeded the in vitro required 90% inhibitory doses on average by two orders of magnitude compared to one order of magnitude in nonresponders. Similar levels of antibody were required to protect from HIV challenge in animal models 6,7,11,12. In contrast, comparably high ratios between in vivo and in vitro antibody doses or pronounced differences between responders and nonresponders in these ratios were not found for monoclonal antibodies 2F5 and 4E10. It thus remains possible that in vivo concentrations of 2F5 and 4E10 had been too low to control viremia and to exert a selective pressure. Whether and why these antibodies were indeed ineffective in vivo will require further investigations in different settings. Several aspects may account for the high antibody doses required for inhibition in vivo compared to those that are effective in vitro. In vitro activity of neutralizing antibodies is traditionally determined by evaluating the activity of antibodies against free virus. But infection through free virus is probably less important than cell-cell transmission in spreading and maintaining viremia in vivo 45. Moreover, cell-cell transmission is considered to be less accessible for inhibition by neutralizing antibodies 46. It is, however, possible that although relatively high antibody doses were required to suppress viremia in established infection, lower antibody doses may suffice to protect from de novo infection in which free virus infection may be more prominent. How well the intravenously delivered antibodies dissipated into the tissue, NATURE MEDICINE ADVANCE ONLINE PUBLICATION 5

6 whether dissemination differed between the individual monoclonal antibodies and what the local concentrations were at the relevant sites of replication could not be addressed in this study, but may be key to the understanding of the inhibition process in vivo. Collectively our data provide direct evidence that antibodies have the capacity to contain viremia in established human HIV-1 infection. But they must be potent and present at high doses to be effective. Moreover, as shown previously 29,31, immune pressure by neutralizing antibodies was subject to rapid escape, suggesting that a successful immunogen will probably have to elicit potent responses to multiple epitopes to limit viral evolution. It is difficult to ascertain in the human setting whether activity of the antibodies was solely through direct virus neutralization or mediated also by effector mechanisms (complement, antibody-dependent cellular toxicity). These questions notwithstanding, our finding that a specific and potent humoral immune response is capable of controlling viremia over extended time periods is of specific relevance for our understanding of the interplay between HIV and host immune responses and underline both the potential and limits of humoral immunity in controlling HIV-1 infection. Although other dosages may apply for protection against infection, our data show that in therapeutic intervention humoral immune responses must be exceptionally broad and potent to be effective. METHODS Subjects. Subject demographics and criteria for enrollment are listed in Table 1, Supplementary Table 1 and Supplementary Note online. We enrolled six acutely infected and eight chronically infected patients in a prospective, nonrandomized and unblinded trial of passive immunization. We recruited all acutely infected subjects for the passive immunization trial or the control group from individuals that participated in a prospective study on ART of early HIV infection. Chronically infected individuals were recruited from a group of individuals who had previously received ART but at the time of blood sampling for prescreening had undergone treatment interruption in clinical trials at our center 42,43. All individuals enrolled in the study in both intervention and control groups had clinical stage A infection. We selected subjects for the passive immunization trial from 27 acutely and 31 chronically infected individuals based on the sensitivity of their autologous virus isolates to the monoclonal antibodies 2G12, 2F5 and 4E10 (ref. 41). Approval of the ethical committee and written informed consent from all subjects were obtained according to the guidelines of the University Hospital Zurich. Study design. Monoclonal antibodies 2G12, 2F5 and 4E10 have been described previously Antibodies were produced by recombinant expression in CHO cells as IgG1(κ) as described We administered monoclonal antibodies sequentially as approximately 10 mg/ml concentrated buffered aqueous solutions starting with monoclonal antibody 2G12, followed by 4E10 and 2F5. Antibody doses in each infusion were 1 g for 2G12 and 4E10 and 1.3 g for 2F5 to compensate for the shorter half-life of this antibody At the first immunization monoclonal antibodies were infused over 1 h each; subsequent infusions were administered in 30-min intervals. The first infusion was given 1 d before cessation of ART, a second infusion 3 4 d later and a third infusion after 7 d, followed by further infusions in weekly intervals up to week 11. In total, each subject received 13 infusions of each monoclonal antibody. Pharmacokinetic analysis of 2F5, 4E10 and 2G12 concentrations in plasma. We quantified plasma concentrations of 2F5, 4E10 and 2G12 using previously established 2F5-, 4E10- and 2G12-specific double-sandwich enzyme-linked immunosorbent assays (ELISA; limit of detection, 3 ng/ml) During the antibody administration phase, levels of monoclonal antibodies were measured before antibody infusion and 30 min after the infusion of Figure 3 Antibody escape. 50%, 70% and 90% inhibitory doses (ID) of 2F5, 4E10 and 2G12 against subject isolates derived before the initiation of the passive immunization trial (pre) and at sequential time points during passive immunization. Numbers on the x-axis indicate the week of the trial when virus was isolated. 50%, 70% and 90% IDs are depicted by open, hatched and full bars, respectively. Violet, blue and orange indicate inhibitory doses of 2F5, 4E10 and 2G12, respectively. Data shown are means of 2 5 independent inhibition assays. 6 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

7 Figure 4 Response analysis. (a e) Subjects were grouped as nonresponders (open symbols; NAB01, NAB02, NAB05, NAB06, NAB07, NAB09, NAB10, NAB11) or responders (closed symbols; NAB03, NAB04, NAB08, NAB12, NAB13, NAB14) and characteristics of the groups were compared using Mann Whitney test. Solid bars indicate median values. 2F5, 4E10 and 2G12 are depicted with violet, blue and orange symbols, respectively. (a) Pre-ART viral loads (RNA copies/ml plasma; log). (b) CD4 cell count (cells/µl blood) on day 0 of the passive immunization trial. (c) Comparison of mean trough levels of monoclonal antibody between weeks 1 and 12 in responders and nonresponders. (d) Comparison of in vitro 90% inhibitory doses of monoclonal antibodies against the latest pre-art isolate. Note: isolates from NAB09 and NAB14 were not inhibited by 2G12 at levels below 25 µg/ml. For analysis, ID 90 levels of these monoclonal antibody virus combinations were set arbitrarily as 25 µg/ml. P value after exclusion of patients with ID 90 > 25 µg/ml is P = (e) Comparison of ratio between mean trough levels of monoclonal antibody between weeks 1 and 12 and respective ID 90 levels. P value after exclusion of subjects with ID 90 > 25 µg/ml is P = the last antibody to determine trough and peak concentration of the antibodies, respectively. During the observation phase between weeks 12 and 24, antibody levels were determined in biweekly intervals. Individual plasma concentrationtime data comprising the samples collected during the first dosing interval, the steady-state trough levels (weeks 3 11), and all concentrations after the last dose (weeks 11 24) fitted well to a two-compartment intravenous infusion model 47 (range, r 2 = ). Distribution half-life (t 1/2α ) and elimination half-life (t 1/2β ) were calculated by dividing ln(2) by the distribution and elimination rates, respectively. Quantification of plasma viral load. We quantified plasma HIV RNA by using the Amplicor HIV-1 Monitor test, version 1.5 (Roche Diagnostics, Rotkreuz, Switzerland) with an ultrasensitive modification 48. Stimulated primary CD8-depleted peripheral blood mononuclear cells (PBMC). Buffy coats obtained from three healthy blood donors were depleted of CD8 + T cells using Rosette Sep cocktail (StemCell Technologies Inc.) and PBMC isolated by Ficoll-Hypaque centrifugation. Cells were adjusted to /ml in culture medium (RPMI 1640, 10% FCS, 100 U/ml interleukin-2, glutamine and antibiotics), divided into three parts and stimulated with either 5 µg/ml phytohemagglutinin, 0.5 µg/ml phytohemagglutinin or CD3-specific monoclonal antibody OKT3 as described 49. After 72 h, we combined cells from all three stimulations and used them as source of stimulated CD4 + T cells for infection and virus isolation experiments. a c d e b Autologous patient viruses. We isolated autologous virus from subjects PBMC by co-culturing subjects CD4 + T cells with stimulated CD8-depleted PBMC. Virus was isolated in absence of the neutralizing antibodies to ensure that escape to antibody treatment occurred in vivo and not in vitro. Only early-passage viruses were used for further studies (passages 1 3). The 50% tissue culture infectious dose (TCID 50 ) and coreceptor usage of the obtained virus stocks were determined as described 49. Neutralization assay. Neutralization activity of monoclonal antibodies against sequential virus isolates was evaluated on CD8-depleted PBMC. Virus inoculum (100 TCID 50 ) was incubated with serial dilutions of antibodies for 1 h at 37 C. Then stimulated PBMC were infected with aliquots of this preincubation mixture. The total infection volume was 200 µl. We incubated cultures in 96-well culture plates for 6 14 d and assayed them for p24 antigen production. The antibody concentrations (µg/ml) causing 50%, 70% and 90% reduction in p24 antigen production were determined by linear regression analysis. If the appropriate degree of inhibition was not achieved at the highest or lowest drug concentration, a value of > or < was recorded and these upper or lower limits were used for statistical analysis. Data analysis. Statistical analyses were performed using GraphPad Prism version 4.0, GraphPad Software Inc. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS Support was provided by the Swiss National Science Foundation (grant PP00B to A.T. and grant 3100A to H.G. and A.T.), research grants from the Union Bank of Switzerland, the Gebert-Rüf foundation (P-041/02) and the FAIR Foundation to A.T. and H.G., and by a research grant of the Kanton Zürich. We thank our patients for their commitment, M. Winniger, U. Berberat, R. Hafner, B. Hasse, U. Karrer, R. Oberholzer, C. Schneider and C. 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