Primary HIV-1 infection is characterized by an uncontrolled viraemia, which often reaches peak plasma titres > 500 000 copies/ml . After seroconversion, this titre drops to a lower steady-state level, the viral ‘set point', which is a prognostic indicator for the subsequent rate of disease progression [2–5]. Several studies have reported the detection of cytotoxic T lymphocyte activity concurrent with this initial decrease in plasma viral RNA levels [6,7], suggesting that the control of plasma viraemia is partly through cell-mediated immune responses. In contrast, neutralizing antibodies are generally not detectable until several months later [6,8–10]. A similar delay in neutralizing antibody production has been reported for SIV infection in macaques [11,12].
Early reports determined neutralizing activity of plasma samples using unrelated viruses such as T cell line adapted laboratory strains or heterologous primary isolates [13–17]. This limited the neutralization activity measured to a cross-reactive component. Autologous isolates are clearly a better choice. However, even limited peripheral blood mononuclear cells (PBMC) co-culture may favour the selection of faster growing viruses that adapt well to in vitro growth but may not necessarily be good representatives of the viruses present in vivo [18–21]. Also, when using viral quasispecies, or ‘swarms', it is common to see neutralization curves plateau at a level below 90%, suggesting that minor populations of neutralization-resistant viruses may obscure detection of some neutralizing antibodies [22,23].
As the envelope is the primary target for HIV-1 neutralizing antibodies, and since an early neutralizing activity may be highly type specific, we wished to address the role of antibodies in acute infection using recombinant viruses bearing envelopes directly isolated from autologous patient PBMC. Using this approach in a single-patient study, we have previously reported that autologous neutralization coincided with a 2 log10 copies/ml reduction in viral load at 13 days after onset of symptoms .
Here we report development of autologous neutralizing antibodies, as assayed against sequential chimeric viruses with autologous envelopes, in four males who contracted HIV-1 through homosexual contact. In contrast to our expectations , no early neutralization was observed in these patients. The patients first developed autologous neutralizing antibodies after termination of primary viraemia at 3, 5, 7 and 16 months after onset of symptoms, respectively.
Interestingly, we could detect ‘non-neutralizing’ envelope antibodies within 20 days of onset of symptoms in all patients. Since our in vitro assays are done in the absence of complement and without the cells required for antibody dependent cellular cytotoxicity, we cannot rule out that these ‘non-neutralizing’ antibodies may contribute to the initial reduction of viraemia in vivo.
The development of neutralizing antibody responses was monitored in four men (MM1, MM2, MM4 and MM8) who contracted HIV-1 through homosexual contact. Acute HIV-1 infection was diagnosed by the detection of HIV-1 genomes (PBMC proviral DNA or plasma RNA) in the presence or absence of an evolving HIV-1 antibody profile that subsequently became fully positive, or by a fully positive HIV-1 antibody test within 3 months of a negative HIV-1 antibody test (MM4). All subjects declined antiretroviral therapy and remained treatment naive throughout the study. Blood samples were obtained weekly for the first month, monthly for 3 months and then at 3-monthly intervals. At each visit, the patients’ HIV-1 viral load (Chiron 3.0; Chiron, Emeryville, California, USA) and CD4 cell count were determined. The study protocol was approved by the Camden and Islington NHS Trust Ethics Committee and written informed consent obtained from all subjects.
Cloning of gp120
Proviral DNA was extracted from whole blood samples preserved with ethylenediaminetetraacetic acid using the QIAamp DNA Blood Mini kit (Qiagen, Cologne, Germany), in accordance with the manufacturer's instructions. Full-length gp120 sequences were amplified by ‘nested’ polymerase chain reaction (PCR). For each patient, the gp120 region of HIV-1 was amplified from a sample collected around the time of symptomatic primary HIV-1 infection and from a second time-point 2–8 weeks later. The PCR was performed using the Expand Long Template PCR system (Roche Molecular Systems, Branchburg, New Jersey, USA) in 50 μl volumes containing 1× Expand buffer 3 (2.25 mmol/l Mg2+), 2.5 U proofreading ‘Expand'–polymerase mixture, 400 μmol/l each of the four dNTP, 200 nmol/l of each primer and patient DNA equivalent to 5–10 μl blood content (> 4000 proviral copies). For the first round of amplification, primers 627L (5′-GATGTTGATGATCTGTAGTGC-3′), 989L (5′-TCATCAAGTTTCTCTAYCAAAGC-3′), 632L (5′-GCGCCCATAGTGCTTCCTGCTGC-3′), and 631L (5′-CCAGACTGTGAGTTGCAACAGAT GC-3′) were used; the inclusion of alternative primers allowed for PCR amplification even when one of the primers failed to match the template sequence. The PCR consisted of 30 cycles of 92°C for 45 s, 45°C for 45 s and 68°C for 210 s. From the first PCR products, 2 μl samples were subsequently re-amplified in 20 μl reaction volumes containing primers 944S (5′-AGAAAGAGCGGCCGCCAGTGGCAATG-3′, start codon bold) and E400010 (5′-GGAGAATTCTTA CCACTGCTCTTTTTTCTCTCTGCACCACT-3′, synthetic stop anticodon bold). The second PCR reactions consisted of 25 cycles of 92°C for 35 s, 55°C for 35 s, and 68°C for 150 s. The PCR products were cloned into the pCR3.1 vector (Invitrogen, Carlsbad, California, USA) and sequenced (Big Dye Terminator Kit, ABI, Foster City, California, USA).
Generation of recombinant viruses with autologous envelopes
Replication-competent viruses were generated by transferring the patient-derived gp120 sequences into the pHxB2-MCS-Δ-env vector previously described . This vector allows incorporation of heterologous gp120 sequences from amino acid 38 (seven amino acids after the signal peptide) to six amino acids prior to the gp120/gp41 junction. Selected gp120 clones were reamplified with the primer pair 626L (sense) 5′-GTGGGTCACCGTCTATTATGGG-3′) and 125Y (antisense) 5′-CACCACGCGTCTCTTTGCCTTG GTGGG-3′, which contain BstEII and MluI sites (bold), respectively, and cloned into the pHxB2-MCS-Δ-env vector. The pHxB2/pg120 clones were confirmed by DNA sequencing. Viruses were produced by transfecting 293T cells with the chimeric constructs and harvested after 72 h.
Virus titration and coreceptor typing
Tenfold serial dilutions of viral stocks were plated on NP2/CD4/CCR5 or NP2/CD4/CXCR4 indicator cell lines seeded in 48-well trays the previous day (2 × 104 cells/well). The cells were incubated with the virus for 2 h at 37°C, washed once in Dulbecco's Modified Eagle Medium (DMEM), overlaid with growth medium [DMEM supplemented with 5% (v/v) fetal calf serum, 1 μg/ml puromycin and 100 μg/ml G418] and cultured for 72 h. Infection was detected by p24 immunostaining, as previously described . Briefly, fixed cells were incubated with mouse anti-HIV-1 p24 monoclonal antibodies (ADP 365 and 366, NIBSC, UK; 1:40 dilution for 1 h) followed by goat anti-mouse immunoglobulin antibody conjugated to β-galactosidase (Southern Biotechnology Associates, Birmingham, Alabama, USA; at 2.5 μg/ml for 1 h) and incubated with β-galactosidase substrate solution (0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-galactopyranoside in phosphate-buffered saline containing 3 mmol/l potassium ferricyanide, 3 mmol/l potassium ferrocyanide and 1 mmol/l magnesium chloride). Infected cells appear blue and focus-forming units (FFU) were counted microscopically. Viral titres are expressed as FFU/ml.
Virus neutralization by heat-inactivated autologous or heterologous sera was assessed by measuring infectivity reduction. Virus (100 FFU) was incubated with twofold serial dilutions of sera (from 1:10), or medium only, in a total volume of 100 μl at 37°C for 1 h. Virus/serum mixtures were subsequently incubated with target cells (NP2/CD4/CCR5 for chimeric viruses and NP2/CD4/CXCR4 for the T cell line adapted laboratory strain IIIB) in 48-well trays for 2 h at 37°C; after which, the cells were washed once and fresh growth medium added. After 3 days, the cells were fixed and stained for p24 antigen (above). Neutralization titres were defined as the reciprocal of the highest serum dilution giving a 90% reduction (IC90) or 50% reduction (IC50) of infectivity (FFU) compared with medium or a non-immune serum. Each serum sample was assayed in triplicate on at least two occasions with reproducible results.
Expression of gp120 and detection of anti-gp120 antibodies in patient sera
293T cells were transfected with the pCR3.1/gp120 clones. To enhance the level of envelope expression, the cells were infected with a T7 RNA polymerase recombinant vaccinia virus (vTF7-3; American Tissue Culture Collection No. VR-2153) 2 h prior to transfection . Envelopes present in the culture medium (clarified by centrifugation at 2000 × g for 5 min) were harvested 72 h after transfection.
For detection of anti-gp120 antibodies in sera, patient-derived or HIV-1IIIB (MRC AIDS directive-product No. EVA657) gp120 was bound to D7324-coated (10 μg/ml overnight) 96-well Maxisorb plates (Nalgene, NUNC International, Hereford, UK) that had been preblocked with 1% milk powder (Marvel); D7324 (Aalto Bio Reagents, Dublin, Ireland) is a sheep polyclonal autologous antibody pool recognizing a conserved epitope (amino acid sequence APTK AKRRVVQREKR) in the C-terminus of HIV-1 gp120, which is encoded by the antisense primer used in the PCR. Antigens were added at saturating concentrations in 1% Marvel in triethanolamine-buffered saline (TBS), and the plate incubated at room temperature for 2 h. After four washes with TBS containing 0.05% Tween 20 (TBS-T), sera diluted 1 : 100 in 100 μl TMT/GS (4% Marvel, 10% goat serum in TBS-T) was added to duplicate wells with captured envelope and to duplicate wells containing no gp120 (blanks), for background subtraction. After six washes with TBS-T, antibodies were detected with enzyme-linked immunosorbent assay (ELISA) using alkaline phosphatase-conjugated goat-anti-human immunoglobulin antibody (Harlan SeraLab, Crawley Down, UK; diluted 1 : 2000 in TMT/GS) followed by a substrate solution (Lumi-Phos Plus, Aureon BioSystems, Vienna, Austria). The plates were incubated with the substrate in darkness for 1 h and relative light units were measured at 405 nm. Data are presented as an average of blank-corrected readings. For determination of end-point titres, two- or threefold dilution series of sera were tested from 1 : 100. End-point titres were defined as the highest dilution giving a reading of 100 light units above background. At this cut of value, none of the six none-immune control sera tested scored positive at a dilution of 1 : 100.
Patients and samples
Four homosexual men (MM1, MM2, MM4 and MM8) with symptomatic primary HIV-1 infection were studied. MM1 was 44 years old and presented 2 weeks after the onset of an influenza-like illness associated with fevers, myalgias, a generalized rash, a sore throat, mild lymphadenopathy, anorexia and diarrhoea. These symptoms lasted for 1 week. He described a high-risk exposure incident 29 days prior to presentation. MM2 was 31 years old and presented with a 4-day history of fevers, night sweats, lethargy, a sore throat, frontal headaches, generalized lymphadenopathy, anorexia and diarrhoea. His most likely high-risk exposure incident was 12 days prior to presentation. MM4 was 41 years old and presented with a 17 day history of fevers, night sweats, lethargy, a sore throat, cervical lymphadenopathy, anorexia and intermittent diarrhoea. He described several high-risk exposure incidents in the preceding 3 months, the most recent 35 days prior to presentation. MM8 was 33 years old and was admitted to hospital with a 3-day history of fevers, lethargy, myalgias, nausea, vomiting, frontal headaches, generalized lymphadenopathy and diarrhoea. His last high-risk exposure incident was 21 days prior to presentation.
Acute HIV-1 infection was confirmed by detection of proviral DNA in blood in the absence of detectable antibodies (MM2), or in the presence of an evolving HIV-1 antibody profile (MM1 and MM8). All three patients subsequently became fully HIV-1 antibody positive. MM4 was HIV-1 antibody positive in all tests at presentation but had a negative HIV-1 antibody test less than 3 months previously. The patients were monitored through seroconversion and at 3-monthly intervals thereafter. Blood samples were collected at each visit. All individuals remained antiretroviral therapy naive throughout the duration of the study apart from MM2, who commenced therapy at month 26; the final sample described for this individual was, therefore, at month 22.
To monitor the development of anti-envelope antibodies and to study antibody-mediated neutralization, autologous envelopes (gp120, SU) were cloned directly from patient PBMC at the earliest time-point available, usually corresponding to the first sample where primary HIV-1 infection was confirmed (28, 18, 17 and 12 days after onset of symptoms for MM1, MM2, MM4 and MM8, respectively). Later envelope clones were also obtained, isolated on day 84, 32, 52 and 32, respectively (Table 1).
Sequencing and phylogenetic analysis
Sequencing of replicate clones demonstrated that the viral envelope populations were largely homogenous at single time-points (Fig. 1). To assess how representative the cloned envelopes were of the bulk viral population, length polymorphism of the V1/V2 region was assessed by size fractionation of uncloned viral/proviral (reverse transcriptase)-PCR products; this showed that most of the clones were representative of the major envelope species circulating at the time of sampling for all patients (unpublished data). The envelopes isolated from the two time-points from MM2 and MM4 were closely related to each other, whereas the env sequences from the first and second time-points formed distinct clusters in patients MM1 and MM8 (Fig. 1).
Development of anti-envelope antibodies
The development of antibodies to autologous envelopes and to IIIB virus envelopes was assessed by ELISA. Both autologous and heterologous (IIIB) gp120 antibodies were detectable in the first (MM1, MM2 and MM4) or second (MM8) available serum samples. This was coincident with the development of a fully positive HIV antibody profile at 18 and 17 days after onset of symptoms for patients MM2 and MM4, respectively. MM8 was confirmed positive at day 12 by clinical tests but we could first detect anti-gp120 antibodies at 20 days after onset of symptoms. For MM1, we obtained a serum sample from day 14 that, in addition to his day 19 sample (when diagnosis was confirmed), contained detectable level of anti-envelope antibodies. The data for MM1 and MM4 are shown in Fig. 2. For all patients, the levels of anti-envelope antibodies rose steadily and reached a plateau by 1 year. Antibody titres to autologous and heterologous envelopes were similar.
Development of neutralizing antibodies
For the neutralization studies, at least one representative envelope (gp120) from the two time-points for each patient was selected and transferred into an HxB2-based vector to generate replication-competent viruses. The patient-derived envelope sequences began seven amino acids after the signal peptide and ended six amino acids before the cleavage signal of gp120/gp41. The gp41 in these viruses is derived from HxB2. We previously showed that viral envelopes maintain their conformation-sensitive neutralizing antibody-binding sites in this construct . The recombinant viruses were tested for co-receptor use on NP2/CD4/CXCR4 and NP2/CD4/CCR5 cells and all envelopes were categorized as R5 (data not shown).
Development of neutralizing antibodies specific for the recombinant viruses was assessed in an infectivity reduction assay. The neutralization titre is expressed as the reciprocal of the highest serum dilution at which 50% or 90% reduction in viral input was observed (Tables 2 and 3).
MM8 developed autologous neutralizing antibodies earliest, between 2 and 3 months after onset of symptoms (Table 2). With the 90% cut-off criteria, neutralizing antibodies appeared simultaneously to MM8's first envelope from day 12 and second envelope from day 32 (1 : 10). This neutralizing antibody response was sustained throughout the study and peaked at 19 months (1 : 40–1 : 80). The other three patients, MM1, MM2, and MM4, showed no evidence of neutralization for at least 4 months. MM1 was still negative at 10 months and only scored positive from month 16 onwards and with low titres (1 : 10–1 : 20) to envelopes from both time-points (day 28 and 84). MM2 developed neutralizing antibodies at the 5-month sampling (1 : 10), which increased and stabilized after 10 months at relatively high titres (1 : 40–1 : 80). MM2's serum was tested against one single autologous envelope-bearing virus, derived from day 32, as earlier envelope chimerae did not produce high enough virus titres. Since MM2's first and second envelope clones were very similar (Fig. 1), it seems unlikely that inclusion of the earlier envelopes would have altered the date from which MM2 could be scored positive. MM4 was neutralization positive from 7 months (1 : 20) and, interestingly, developed a more neutralization sensitive envelope with time. However, in contrast to the other envelopes used for the neutralization studies, this envelope (4.4.48) was a minor virus variant in vivo. The V1/V2 region of the 4.4.48 envelope is one amino acid longer than other envelopes cloned from MM4, and this length formed a only a minor peak in length polymorphism studies of uncloned time-matched proviral DNA.
As our results were discordant with our own earlier observation  and that of others [28,29], who reported the detection of autologous neutralizing antibodies within a couple of weeks of seroconversion, our data were re-evaluated using a less stringent 50% cut-off (IC50, Table 3). By this criterion, patients MM2 and MM8 scored positive at earlier time-points: 3 months (compared with 5) and less than 3 weeks (compared with 3 months), respectively. This earlier neutralizing activity in MM8 was detectable with the first time-point (day 12) envelopes. MM1 was positive at 2 months, but this neutralizing activity disappeared at the 3-month time-point, reappearing at month 16 when 90% neutralization could be detected. Therefore, even with the lower stringency of 50%, only one patient (MM8) could be clearly recategorized as having developed early autologous neutralization that could potentially have contributed to the termination of primary viraemia (see below). However, in this assay format, a 10–20% reduction of infection with non-immune sera was often observed. On occasion, this could reach 60%, depending on the viral envelope and particular sera, hence a cut-off at 50% was regarded as unreliable and of questionable significance.
Heterologous neutralization of the IIIB isolate was also measured. In contrast to autologous neutralization, 90% inhibition of IIIB was only observed in one patient, MM2, who scored positive from month 13 onwards, 8 months after the first detectable autologous neutralizing activity. His neutralizing antibody titre against IIIB was substantially lower than his autologous titre (1 : 10 compared with 1 : 80). Using IC50 for IIIB, all four patients scored titres up to 1 : 20. However, this heterologous neutralizing activity remained weak and sporadic throughout the study period.
To summarize, despite the rapid development of antibodies that bound autologous envelope, neutralization was not observed until after 3 months after onset of symptoms in three of the four patients described here. There was a large variation between the patients in the time to first detection of autologous neutralizing antibodies: 3 (or 20 days for 50%), 5 (or 3 month for 50%), 7 and 16 months. Heterologous virus neutralization was delayed by at least 3 months and remained weak, again in contrast to the early development of antibodies that bound the heterologous envelope.
As shown above, the development of heterologous neutralization in these patients was transient and delayed compared with autologous neutralization. All viruses were assessed for their relative sensitivity to neutralization using a panel of unrelated HIV-1- positive sera (Table 4) . As expected, IIIB was more sensitive than the primary envelope chimerae, at least to neutralization by polyclonal human serum. Therefore, most neutralizing antibodies were directed towards early autologous virus and their reactivity broadened with time. Interestingly, the clone from the second time-point from MM4 (4.4.48) was as sensitive to neutralization as IIIB. This sensitivity was also apparent with sequential autologous sera. Sequencing of 4.4.48 revealed that this relative sensitivity to neutralization was associated with major changes from the earlier less-sensitive envelopes from the same patient. These changes included one truncation (3 amino acid residues), two insertions (2 amino acid residues each) and 16 amino acid substitutions in the V1/V2 loop together with single amino acid substitutions in C1 and V5 (unpublished data).
Collated data for antibody levels, plasma viral load and CD4 cell counts are presented in Fig. 3. The time-point for the development of autologous neutralization is also indicated. Patients MM1, MM4, and MM8 demonstrate ‘typical’ seroconverter profiles with regard to HIV-1 viral load and CD4 cell count. Their viral load was already in decline at the first sampling point and coincided with a partial recovery of CD4 cell counts, followed by a steady reduction. MM2's CD4 cell count fluctuated at a low level throughout the study. The development of neutralizing antibodies did not correlate with the decline in viraemia for any patient nor did they appear to have any striking effect on viral loads after ‘set point’ was established.
We show here that the development of autologous neutralizing antibodies varies greatly between patients, being detectable from 3 to 16 month after onset of symptoms. Our results agree with previous findings using autologous isolates selected for growth in PBMC [8–10,31–33]. Therefore, direct isolation of viral envelopes without in vitro passage does not alter the time to development of detectable neutralizing antibodies levels following HIV-1 infection. Our findings are also in agreement with two recent papers using a similar approach for detection of neutralizing antibodies with directly isolated early autologous envelopes [34,35]. In both of these studies, development of neutralizing antibodies was detected between 4 and 8 weeks after onset of symptoms in some patients, but also significantly later (more than 36 months) in others .
As expected, no difference was observed in the time for development of neutralizing antibodies to the envelopes derived from the two time-points examined. The establishment of neutralizing antibody escape variants in vivo has been reported, but generally several months after induction of neutralization [28,29,34–36]. Our study was confined to early envelopes derived from viruses circulating before the development of any detectable neutralizing antibodies.
If the stringency of the neutralization assay was reduced to 50% inhibition, one patient (MM8) out of four could be categorized as having developed autologous neutralizing antibodies near seroconversion. Another (MM1) developed an early (within 2 months) neutralizing activity but this response was transient and disappeared within 1 month; the results for MM2 and MM4 were not significantly altered. We propose that the discrepancies between our results and others [28,29] may result from natural cohort variation. Indeed, we have previously reported very early (from 13 days after onset of symptoms) autologous neutralization using this cloning approach in a single-patient study, which prompted the current investigation . A possible explanation for cohort inconsistency is variability in CD4 T helper responses critical for induction, maintenance and maturation of antibody production by B cells. Strong HIV-specific CD4 cell responses have been implicated in control of primary HIV-1 infection . However, all of the patients in this study had weak CD4 T helper responses (unpublished data).
In all cases, heterologous neutralizing antibodies arose later (3 months or more) and to lower titres (IC90 < 1 : 10 to 1 : 20 compared with 1 : 10–1 : 80) than autologous neutralizing antibodies. This, together with the maintenance of neutralizing activity to early autologous virus, suggests a broadening of the immune response with time, as reported by others [10,16,17,33,38].
The development of autologous neutralizing antibodies did not correlate with the decline in primary viraemia in our patient cohort nor did it appear to have any effect on viral loads after ‘set point’ was established, in agreement with other studies [9,10,34]. Interestingly, we observed both heterologous and autologous antibodies arising months prior to the development of neutralizing antibodies, but near maximum levels were reached in all cases before significant neutralization was detected. Perhaps a minimum number of envelope spikes need to be engaged to achieve neutralization, as proposed by Burton et al. . Since our in vitro assays are done in the absence of complement and without the cells required for antibody-dependent cellular cytotoxicity, we cannot, at this stage, exclude that the early low levels of antibodies may have the ability to control viral replication in vivo in the presence of complement [40–42] and/or antibody-dependent cellular cytotoxicity [43–45].
In summary, the study presented here is consistent with previous studies [6,8–10] demonstrating that neutralizing antibodies do not arise prior to the fall in plasma viral load observed in acute HIV-1 infection. The detection of envelope-binding antibodies simultaneously with the reduction in viral load in all patients studied presents the intriguing possibility that other effector functions, besides direct neutralization, may play a role in establishing the viral ‘set point'.
We would like to thank Professor Robin Weiss, Dr Saema Magre and Mr Keith Aubin for their critical reading of this manuscript, and the MRC AIDS Directive and NIBSC for supplying reagents.
Sponsorship: This project was partly funded by the Edward Jenner Institute for Vaccine Research and the MRC and is part of a multicentre collaboration headed by Dr P. Borrow. This is publication number 71 from the Edward Jenner Institute for Vaccine Research. Á. McKnight is funded by the Wellcome Trust (RCDF no. 060758).
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Keywords:© 2004 Lippincott Williams & Wilkins, Inc.
HIV-1; neutralization; autologous; heterologous; seroconversion; antibodies