The ability to elicit potent and cross-reactive neutralizing HIV type 1 (HIV-1)-specific humoral immunity is one of the major goals in HIV-1 vaccine development . One of the current approaches is the characterization of the epitopes of the best broadly neutralizing antibodies (BrNAbs) that are known to date and to use these epitopes as immunogens to elicit HIV-1-specific neutralizing antibodies with similar potency and breadth. However, antibodies elicited by currently available HIV-1 envelope-based immunogens do not display potent and cross-reactive neutralizing activity . Interestingly, the BrNAbs b12, 2G12, 2F5, and 4E10 have all been isolated from HIV-1-infected patients [3–6], suggesting that the native envelopes of the HIV-1 variants in these individuals were capable of eliciting these antibody responses.
Despite intense research efforts, the number of isolated BrNAbs has remained low, which has led to the assumption that BrNAbs are rare in natural HIV-1 infection. However, several recent studies [7–12] have shown HIV-1-specific cross-reactive neutralizing activity in sera from various HIV-1-infected patients. In three studies [7,8,13], the specificity of the neutralizing activity was identified, but it is still unclear whether the breadth of the neutralizing activity in serum is determined by a single high-affinity antibody directed against a highly conserved epitope in the envelope protein, or whether it is the combined effect of multiple coexisting neutralizing antibodies directed at multiple distinct regions of the envelope. In line with both possibilities is the observation that cross-reactive HIV-1-specific neutralizing activity in serum develops over time. Indeed, early in infection, neutralizing activity in serum is directed against autologous HIV-1 variants and rarely directed against heterologous isolates , whereas plasmas collected during the chronic phase of infection display various degrees of cross-reactive neutralizing activities [15–18], although a more exact prevalence of cross-reactive neutralizing activity in sera from HIV-1-infected patients remains to be established.
To support HIV-1 vaccine development, more insight is needed into factors that are associated with the ability of the host to elicit a cross-reactive neutralizing humoral immune response, and how such a neutralizing serum response evolves over time. Here, we studied the potency and breadth of HIV-1-specific neutralizing humoral immunity in serum samples that were obtained at 2 and 4 years after seroconversion from 35 participants of the Amsterdam Cohort Studies (ACS). The prevalence of cross-reactive neutralizing activity in serum in our study group was 31%. We observed a strong correlation between duration of infection and breadth of the neutralizing HIV-1-specific humoral immune response, and a high plasma viral RNA load set-point and low CD4+ cell count set-point were both associated with the early development of cross-reactive neutralizing activity. However, the prevalence of cross-reactive neutralizing activity in serum was similar for long-term nonprogressors (LTNPs) and progressors, excluding a correlation between potent humoral immunity and the clinical course of infection.
Patients and methods
The study group consisted of LTNP (defined as HIV-1-infected patients who have ≥10 years of asymptomatic follow-up with stable CD4+ cell counts that were still above 400 cells/μl in the ninth year of follow-up) and progressors [HIV-1-infected patients who progressed to AIDS within 7 years after (imputed) seroconversion] who were all participating in the ACS on HIV and AIDS in homosexual men. All individuals were infected with HIV-1 subtype B, and were either seropositive at entry in the cohort studies (seroprevalent cases with an imputed seroconversion date on average 18 months before entry in the cohort [19,20]) or seroconverted during active follow-up in the cohort studies. None of the participants received combination antiretroviral therapy during the sampling period; samples were obtained on average at 28 months (range 24–33 months) and 51 months (range 45–83 months) after imputed or documented seroconversion.
The ACS are conducted in accordance with the ethical principles set out in the declaration of Helsinki and written consent was obtained prior to data collection from each participant. The study was approved by the Academic Medical Center institutional medical ethics committee.
U87/pseudovirus assay for testing of HIV-1-neutralizing activity in serum
Sera from all 35 patients were tested for neutralizing activity in a pseudovirus assay developed by Monogram Biosciences. The tier 2–3 virus panel that we used for determining cross-reactive neutralizing activity in serum consisted of HIV-1 pseudoviruses from subtypes A (n = 5), B (n = 6), C (n = 7), and D (n = 5), and included recently transmitted isolates and moderately neutralization sensitive and resistant primary HIV-1 variants, based on previously determined neutralization sensitivities to subtype B sera and mAbs b12, 2G12, and 4E10 [21,22]. Pseudotyped viral particles were produced by cotransfecting HEK293 cells with an expression vector carrying the HIV-1-derived gp160 gene (eETV) and an HIV-1 genomic vector carrying a luciferase reporter gene (pRTV1.F-lucP.CNDO-ΔU3). Forty-eight hours after transfection, pseudovirus stocks were harvested and small aliquots were tested for infectivity using U87 target cells expressing CD4, CCR5, and CXCR4. Pseudovirus stocks were then diluted to result in infectivity, as measured by relative light units that fell within a range known to yield reproducible tissue culture infectious doses (TCID).
A recombinant virus assay involving a single round of virus infection was used to measure cross-reactive neutralization activity of the sera [14,23]. Diluted pseudoviruses were incubated for 1 h at 37°C with serial dilutions of serum after which the U87 target cells were added. The ability of patient sera to neutralize viral infection was assessed by measuring luciferase activity 72 h after viral inoculation in comparison with a control infection with a virus pseudotyped with the murine leukemia virus envelope (aMLV). Neutralization titers are expressed as the reciprocal of the plasma dilution that inhibited virus infection by 50% (IC50). Neutralization titers were considered positive if they were three times greater than the negative aMLV control. 1: 40 was the lowest serum dilution used in the assay. For calculation of IC50 values for viruses that were not inhibited by the 1: 40 serum dilution we assumed that 50% inhibition would have occurred at a 1: 20 serum dilution.
Peripheral blood mononuclear cell-based assay for testing HIV-1-neutralizing activity in serum
Sera from the 19 individuals with a documented seroconversion were tested in parallel in a peripheral blood mononuclear cell (PBMC)-based neutralization assay using both resistant and sensitive tier 2–3 primary HIV-1 variants 92UG029, KNH1144 (subtype A), BX08, BK132 (GS 009) (subtype B), SM145 (GS 016) (subtype C), 92UG038, 93UG065 (subtype D), and CAM1970LE (CRF_AG) . PBMCs were obtained from buffy coats from 10 healthy seronegative blood donors and pooled prior to use. Cells were isolated by Ficoll-Isopaque density gradient centrifugation and then stimulated for 3 days in Iscove's modified Dulbecco medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 U/ml), and phytohemagglutinin (PHA; 5 μg/ml) at a cell concentration of 5 × 106/ml. After inoculation, the cells (1 × 106/ml) were grown in the absence of PHA in medium supplemented with recombinant interleukin-2 (IL-2) (20 U/ml; Chiron Benelux, Amsterdam, The Netherlands) and Polybrene (5 μg/ml; hexadimethrine bromide; Sigma, Zwijndrecht, The Netherlands).
To prevent possible complement-mediated antibody inhibition of virus infection, complement in human sera and fetal bovine serum was inactivated by incubation at 56°C for 30 min. From each virus isolate, an inoculum of 20 50% tissue culture infective doses in a total volume of 50 μl was incubated for 1 h at 37°C with increasing dilutions of the serum (starting concentration 1: 25) in 96-well microtiter plates. Subsequently, 105 PHA-stimulated PBMCs were added to the mixtures of virus and serum. After 4 h of incubation, PBMCs were washed once in 100 μl phosphate-buffered saline after which fresh medium was added. On day 11, virus production in culture supernatants was analyzed in an in-house p24 antigen capture ELISA . Experiments were performed in triplicate. When possible, IC50 were determined by linear regression.
Statistical analyses were performed using the SPSS 16 software package. HIV-1 RNA load in plasma (copies/ml) and CD4+ cell count in blood (number/μl) at set-point were normally distributed and compared between different groups using independent samples t-test. Titer and breadth of the neutralizing activity in serum were not normally distributed and for estimation of correlation coefficients with either viral RNA load in plasma or CD4+ cell count in blood at set-point the nonparametric two-tailed Spearman correlation coefficient was used. Ranking for assay correlation was normally distributed and calculated with Pearson correlation. The Kruskall–Wallis test was used to compare neutralization titers at both years 2 and 4 after seroconversion, per virus, and between patients who had or lacked cross-reactive neutralizing activity in serum. Geometric means of serum neutralization titers were calculated for each patient per time point and the Mann–Whitney test was performed to compare neutralization titers at both years 2 and 4 after seroconversion.
Prevalence of HIV-1-specific cross-reactive neutralizing serum activity
We studied 35 participants from the ACS for the breadth of HIV-1-specific cross-reactive neutralizing activity in sera that were obtained on average 28 months (range 24–33 months) and 51 months (range 45–83) after seroconversion. HIV-1-specific cross-reactive neutralizing activity was measured in a cell-based infectivity assay using recombinant viruses that carry a luciferase reporter gene and that are pseudotyped with envelope proteins from tier 2 HIV-1 subtypes A, B, C, and D. To monitor neutralizing activity not mediated by HIV-1 Env-specific antibodies, each plasma sample was also tested against a recombinant virus stock that was pseudotyped with amphotropic murine leukemia virus (aMLV) envelope proteins (gp70SU and p15TM). Typically, neutralization titers, expressed as the reciprocal of the plasma dilution that inhibited infection by 50% (IC50), were less than 40 for amphotropic murine leukemia virus controls. At 24 months after seroconversion, HIV-specific cross-reactive neutralizing activity, defined as an IC50 at least 100 for at least 50% of viruses per subtype, from at least three different subtypes, was observed in sera from seven individuals (20%, three LTNP, and four progressors; patient IDs indicated in red in Fig. 1). At the 4-year time point, sera from these seven individuals still had high-titer cross-reactive neutralizing activity. Interestingly, at this second time point, high-titer cross-reactive neutralizing serum activity had developed in four additional individuals (three LTNP and one progressor, patient IDs indicated in yellow in Fig. 1) resulting in a prevalence of HIV-1-specific cross-reactive neutralizing serum activity of 31% around 4 years after seroconversion. There was no difference in prevalence of high-titer cross-reactive neutralizing activity in serum between LTNP and typical progressors at either time point of analysis (at year 2 after seroconversion: 15% of LTNP and 27% of progressors; at year 4 after seroconversion: 30% of LTNP and 33% of progressors). Moreover, there were no differences in neutralization titers between LTNP and progressors.
As discrepancies may exist between different neutralization assays , we wanted to confirm our observations obtained with the U87/pseudovirus-based neutralization assay in a PBMC-based neutralization assay. As the primary viruses from which the pseudoviruses were derived were not available, we used a different panel of eight primary HIV-1 variants from different subtypes (A, B, C, D, and CRF_AG) and serum samples that were obtained 2 years after seroconversion from 19 ACS participants in this study with a documented seroconversion.
The overall pattern of neutralization of the eight viruses by the sera from the 19 patients in the PBMC assay is shown in Fig. 2(a) and (b), and IC50s at least 1: 100 are indicated in green. In accordance with our observations in the U87/pseudovirus-based assay, the serum neutralizing activity against subtype B viruses was the strongest. Unlike the U87/pseudovirus assay, in which subtype C viruses were sensitive to serum neutralization, the selected subtype C virus was resistant to neutralization by all but one of the patient sera.
Overall, neutralizing serum titers in the PBMC-based assay were generally lower, reducing the sensitivity to detect the neutralization breadth of the patient sera as compared with the pseudovirus assay. Indeed, in the PBMC-based assay, none of the sera were able to neutralize all HIV-1 variants from all different subtypes. However, a significant correlation between the two assays could be observed when patients were ranked on the basis of neutralization breadth and potency (Fig. 2c). Ranking was assigned by giving priority to serum ability to neutralize different subtypes, followed by the total amount of viruses neutralized and finally by the titers at which the viruses were neutralized.
Correlation between set-point viral load and CD4+ cell count and breadth of HIV-1-specific cross-reactive neutralizing serum activity
Our data indicate that cross-reactive neutralizing serum activity does not develop similarly in the course of infection for each HIV-1-infected patient. To obtain some insight into factors that may influence the humoral immune response, we divided the cohort of 35 patients who participated in our study in three distinct groups: patients who had no detectable cross-reactive neutralizing activity at years 2 and 4 after seroconversion (group A, n = 24; patient IDs in white in Fig. 1), patients who had cross-reactive neutralizing activity already at year 2 after seroconversion (group B, n = 7; patient IDs in red in Fig. 1), and patients who had developed cross-reactive neutralizing between years 2 and 4 after seroconversion (group C, n = 4; patient IDs in yellow in Fig. 1). These three groups were compared for plasma viral load and CD4+ cell count at set-point (Fig. 3a and b).
Interestingly, the presence of cross-reactive neutralizing activity at year 2 after seroconversion (group B) was associated with a higher plasma viral RNA load set-point as compared with the group of patients who had not developed cross-reactive humoral immunity in the first 4 years after seroconversion (group A). There was no significant difference in viral RNA load set-point between groups A and C, nor between groups B and C (Fig. 3a). These observations were reinforced by a significantly lower CD4+ cell count at set-point in patients with cross-reactive neutralizing activity at year 2 after seroconversion (group B) as compared with the CD4+ cell count at set-point in the other two patient groups (groups A and C, Fig. 3b).
Interestingly, an analysis, which included all 35 individual patients who participated in our study revealed a positive correlation between the plasma viral RNA load at set-point and the number of viruses that were neutralized by the serum sample obtained at year 2 after seroconversion (Fig. 3c). We also observed a negative correlation between the CD4+ cell count at set-point and the number of viruses that were neutralized by the 2-year postseroconversion serum sample (Fig. 3d). No such correlations were observed for the serum neutralizing activity at year 4 after seroconversion (data not shown).
Viral RNA load and CD4+ cell count at the time of sampling did not differ between the groups that developed cross-neutralizing reactivity (groups B and C) and the group that did not develop cross-reactivity within 4 years after seroconversion (group A, data not shown).
Titers of HIV-1-specific cross-reactive neutralizing activity in serum increase with duration of infection
With increasing time since seroconversion, we observed an increase in the geometric mean of the neutralizing titers in serum. This could not be explained by the increasing number of patients who developed cross-reactive neutralizing activity over time. Indeed, when we analyzed the three groups as defined above [individuals who did not develop cross-reactive neutralizing activity in the first 4 years after seroconversion (group A), individuals with cross-reactive neutralizing serum activity at year 2 after seroconversion (group B), and individuals who developed cross-reactive neutralizing serum activity between years 2 and 4 after seroconversion (group C)], the increase in the geometric mean of HIV-1 neutralizing titers in serum was observed in each patient group (Fig. 4). Even for sera from individuals who did not develop cross-reactive neutralizing activity in the first 4 years after seroconversion (group A), an increase of the geometric mean of the neutralizing titers was observed over time, although that the magnitude of the increase was less than that observed for the patients who did develop cross-reactive neutralizing activity.
Finally, we observed that in 91% of patients, an increase in geometric mean of neutralizing titers in serum was observed between years 2 and 4 after seroconversion.
We compared 20 LTNPs and 15 progressors for the presence of HIV-1-specific cross-reactive neutralizing activity in serum at years 2 and 4 after seroconversion. Already at 2 years after seroconversion, seven individuals (three LTNPs and four progressors, overall 20%) had potent cross-reactive neutralizing activity in their sera, defined as the ability to neutralize at least 50% of HIV-1 variants per subtype, from three different subtypes, with an IC50 at a serum dilution of at least 1: 100. Interestingly, these seven individuals had a significantly higher set-point viral RNA load in plasma and a lower CD4+ cell count at set-point than individuals who lacked a potent cross-reactive neutralizing response. The development of potently neutralizing humoral immunity apparently requires exposure to a sufficient amount of antigen, in line with previous observations . Alternatively, a better exposure of epitopes on envelope that are essential for eliciting a cross-reactive neutralizing humoral immune response may coincide with enhanced replication kinetics resulting in a higher plasma viral RNA load set-point. In a model for lymphocytic choriomeningitis virus infection, a reduction in CD4+ T cell numbers prior to infection reduced polyclonal B-cell stimulation and enhanced protective antibody responses in terms of earlier onset and higher titers without impairing protective CD8+ T-cell responses [27,28]. Although the number of patients in our study is low, our observation that early cross-reactive neutralizing activity correlated with a low CD4+ cell count at set-point may imply that this could also be the case in HIV-1 infection.
The fact that the majority of primary HIV-1 variants are neutralized by one or more of the currently available broadly neutralizing antibodies b12, 2G12, 2F5, and 4E10, already implies that the epitopes for these broadly neutralizing antibodies are accessible on primary viruses. It is generally assumed, however, that the configuration of the envelope prevents the elicitation of a neutralizing antibody response in vivo. The relatively high prevalence of cross-reactive neutralizing serum activity, which is similar to observations in other studies [8,11], however suggests that the relevant epitopes capable of eliciting these humoral responses are accessible and immunogenic on the native gp160 spike of HIV-1, at least in a significant proportion of HIV-1-infected patients. We may be more conclusive on this point when the exact nature of the neutralizing activity in our study group has been established. Indeed, it is unclear whether the breadth of the neutralizing activity in serum is determined by a single high-affinity antibody directed against a highly conserved epitope in the envelope protein, or whether cross-reactive neutralizing activity in serum can be attributed to a combination of multiple coexisting neutralizing antibodies directed at a number of distinct regions of the envelope that together give the phenotype of a cross-reactive serum neutralization. It cannot be excluded that both scenarios exist and that it may vary between individuals. Interestingly, a recent study by Scheid et al.  has demonstrated the presence of a relatively large memory B-cell repertoire capable of producing different antibody specificities in HIV-1-infected patients with cross-reactive neutralizing serum activity.
Irrespective of the nature of the neutralizing response, the 2–4 years that seem to be required to achieve a potent neutralizing immune response and then only in 31% of patients, at least in our study population, may hamper the efficacy of vaccine-induced humoral immunity. The use of optimal adjuvants may be essential to accelerate the development of broadly neutralizing antibodies after immunization. However, several studies [30–32] have suggested that low levels of neutralizing titers may actually be sufficient to achieve protection from infection. These lower titers may be achieved more rapidly than the 1: 100 serum dilution threshold we set for our experiments shown here.
How HIV-1 neutralizing activity in vitro relates to protection from infection in vivo , which may be better reflected in a PBMC-based neutralization assay remains to be established. However, our initial data on the cross-reactive neutralizing activity in sera obtained with a pseudovirus-based assay on U87 cells were confirmed in a PBMC-based assay using replication competent primary HIV-1 variants from different subtypes. Simek et al.  have demonstrated that neutralizing activity can indeed be reliably assessed using pseudovirus panels. The ability of our patient sera to neutralize viruses from the large virus panel that we used in our present study was strongly correlated with the ability of these same sera to neutralize viruses from the seven virus panel used by Simek et al.  (Spearman r = 0.77, data not shown) not only confirming the validity of the large virus panel that we used but also strengthening our conclusion on the broadly neutralizing ability of the patient sera tested in our study.
Although the number of patients in our study is relatively small, our data suggest that there is no correlation between the presence of cross-reactive neutralizing activity in serum and the clinical course of infection. Indeed, we observed a similar prevalence of cross-reactive neutralizing serum activity at 2 and 4 years after seroconversion in LTNPs and progressors. Moreover, the presence of cross-reactive neutralizing antibodies in serum did not coincide with a reduction in viral load, in line with the observation that administration of broadly neutralizing antibodies to human peripheral blood leukocytes (hu-PBL)-severe combined immune deficiency (SCID) mice after inoculation with HIV-1 had no effect on viral load in the animals .
Our data are supportive for the idea that it is important to achieve vaccine elicited sterilizing immunity that prevents establishment of infection, or a vaccine that can elicit potent cross-reactive neutralizing humoral immunity in combination with effective cellular immunity to delay or prevent disease progression . The relatively high proportion of individuals with cross-reactive neutralizing humoral immunity in our present study and other studies [8,11] suggests that the B-cell repertoire in humans should indeed be sufficient to respond to a vaccine with potently neutralizing antibodies implying that a protective antibody-based vaccine against HIV-1 may be an obtainable goal.
The ACS on HIV infection and AIDS – a collaboration between the Amsterdam Health Service, the Academic Medical Center of the University of Amsterdam, Sanquin Blood Supply Foundation, and the University Medical Center Utrecht – are part of The Netherlands HIV Monitoring Foundation and are financially supported by The Netherlands National Institute for Public Health and the Environment. The research leading to these results has received funding from the European Community's Six Framework Programme Europrise (FP6/2007-2012) under grant number 037611, the European Community's Seventh Framework Programme NGIN (FP7/2008-2012) under grant agreement no. 201433, The Netherlands Organisation for Scientific research (NWO; grant #918.66.628) and NIH Small Business Innovation Research (SBIR) grant (#5R44AI062522) awarded to Monogram Biosciences. The funding organizations had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: 92UG029, KNH1144, CAM1970LE, BX08 from Dr Victoria Polonis, BK132 (GS 009), SM145 (GS 016) from Dr Nelson Michael, 92UG038, and 93UG065.
H.S. designed the study and supervised all aspects of the study. T.W. and B.S. designed, performed, and analyzed the pseudovirus panel neutralization experiments. Z.E. performed the PBMC neutralization experiments. M.J.G. and Z.E. analyzed and interpreted the data and prepared the manuscript. All authors contributed to the writing of the manuscript and approved the final version.
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