Identification of HIV sequences recognized by early-phase seroconversion sera using GFPDL.
To identify all the HIV sequences recognized by antibodies generated soon after HIV infection, we constructed a GFPDL spanning the entire HIV-1 open reading frame of NL4-3. The HIV-1 GFPDL contained >107 independent transformants. PCR-based analysis and sequencing of the inserts confirmed that the library consisted of 100% recombinants, with insert sizes of 50 to 300 bp and random distribution across the HIV genome.
Seven plasma samples constituting seroconversion panel PRB-910 (obtained from acutely HIV-1-infected individuals; SeraCare BioServices, Gaithersburg, MD) were used for affinity selection of phages displaying HIV-1 peptides. After four rounds of affinity selection, 22 clones (for each plasma sample) were selected for insert sequencing and were reanalyzed by phage ELISA with other panels of HIV-positive and -negative sera to confirm the specificity of reactivity. An alignment of inserts with the HIV-1 genome led to the identification of 12 immunodominant epitopes, mapping to Gag (p24 and p6), Pol, envelope (gp120 and gp41), and Nef. Interestingly, phages displaying sequences from the intracytoplasmic tail of gp41 (amino acids 784 to 871) were repeatedly recognized by antibodies from both acutely (1 to 6 months) and chronically HIV-infected individuals. The cytoplasmic tail of gp41 was selected as the primary candidate for the differential assay because it is unlikely to be targeted by HIV-neutralizing antibodies and is not included in most HIV vaccines currently under development. In addition, a p6 sequence was selected, even though it was included in some early HIV vaccines, since it contains very few HLA-restricted cytotoxic T-lymphocyte epitopes (10
) (Los Alamos database [http://hiv-web.lanl.gov
]). Importantly, the selected gp41 (amino acids 784 to 829 [SK1] and 836 to 871 [SK2]) and p6 (amino acids 452 to 502) sequences are highly conserved among all HIV-1 M subtypes.
Establishment of HIV-SELECTEST.
The p6- and gp41-derived peptides were chemically synthesized and used for the development of the new assay. Peptides were designed based on the consensus sequences representing HIV-1 group M subtypes (Los Alamos HIV sequence database) to encompass the genetic variability among HIV-1 clades. Initially, each peptide was evaluated individually to determine its specificity and to establish cutoff values. Since both gp41 peptides (SK1 and SK2) displayed similar very low reactivities with HIV-seronegative samples, the two were combined. Multiple ELISA conditions were tested, and after screening of 1,000 seronegative samples, cutoff (CO) values for the gp41 (CO = 0.03) and p6 (CO = 0.15) peptides were determined. Each CO value represents the average absorbance value for negative sera (at a 1:100 dilution) plus 5 standard deviations. Additional serum panels containing high, intermediate, and low HIV-specific antibody titers were used to determine the dynamic range of the assay. Panels a and b in Fig. demonstrate the binding of a serially diluted representative HIV-1-positive plasma, PRB-204-06 (from SeraCare BioServices), in the p6 and gp41 ELISAs, respectively. Titrations with additional samples demonstrated a higher maximum reactivity with the gp41 peptides and a broader dynamic range than those with the p6 peptide. Based on these analyses, all subsequent ELISA testing was conducted with a 1:100 dilution of serum or plasma. The HIV infection status of a given sample was determined with licensed detection kits conducted in-house and/or by other laboratories. Assay specificities of 100% for the gp41 peptides and 99.4% for the p6 peptide were established after screening of >2,500 samples either from uninfected individuals or from individuals infected with diverse HIV-1 clades. The combined sensitivity of the gp41 and p6 peptides was 99.3% for the detection of recent and chronic HIV infections in multiple geographical sites with clades A, B, C, D, E, F, and J and circulating recombinants (manuscript in preparation).
FIG. 1. Dynamic range of serum reactivities and reproducibility of the p6 and gp41 peptide-based HIV-SELECTEST. The ELISA conditions used are described in Materials and Methods. A well-characterized panel of nine HIV-seropositive and three HIV-seronegative human (more ...) Assay robustness and statistical analysis.
The reproducibility of the assay was determined by repeatedly testing nine HIV-seropositive and three HIV-seronegative samples from SeraCare BioServices. The distributions of the results obtained on multiple dates were evaluated for normality, and the appropriate P values were calculated using SigmaPlot. Representative plots are shown for one individual for the p6 and gp41 peptides (Fig. , respectively). The upper and lower limits (±2 SD) represent the 95% confidence intervals. Interassay variability was ≤10%, and intraassay variability was ≤5% for all samples tested.
Acute infections are detected with HIV-SELECTEST.
To determine how soon postinfection HIV-specific antibodies are detected with the HIV-SELECTEST, several well-characterized seroconversion panels were obtained from SeraCare BioServices containing sequential bleeds taken within 10 to 40 days of estimated exposure dates. As shown in Table , the p6 peptide reacted positively with PBR-910 on collection day 26, in agreement with results obtained using licensed HIV antibody detection kits. The gp41 peptides were reactive with the day 32 sample from the same individual. For PRB-929, day 25 and day 28 samples reacted with the p6 and gp41 peptides, respectively (Table ). For that individual, infection was confirmed by PCR on day 14, and the Abbott HIV Ag test was positive on day 18. Similar results were obtained with additional seroconversion panels from SeraCare BioServices (data not shown) and demonstrated that HIV infection could be detected by the HIV-SELECTEST within 2 to 4 weeks following HIV-1 RNA detection by PCR, concurrent with the sensitivity limits of licensed HIV diagnostic tests. In addition, we evaluated 28 HIV seroconversion panels from Australia spanning 6 to 18 months postinfection (Table and data not shown). With these panels, p6 showed variable reactivities at later times postinfection, whereas anti-gp41 reactivity increased over time and was maintained at high levels in most individuals, indicating that the kinetics and avidity of the antibody responses against the p6 and gp41 epitopes were not linked.
Early detection of HIV-1 infection by HIV-SELECTEST with seroconversion panels
Detection of early HIV-1 infection by HIV-SELECTEST
Evaluation of samples from HIV vaccine trials.
The main proof of concept in support of the HIV-SELECTEST should come from evaluating the reactivities of vaccine-induced antibodies in the course of prophylactic vaccine trials. To that end, six blinded panels from completed vaccine trials (502 vaccinees), including HVTN 203 (conducted by the HIV Vaccine Trial Network), RV124 (conducted by the Walter Reed Army Institute of Research), VRC 004, VRC 006, VRC 009, and VRC 010 (conducted by the Vaccine Research Center, NIAID, NIH), were tested. A description of the vaccine constructs used in the various trials and a summary of the results obtained with the HIV-SELECTEST appear in Table . Data are shown for samples tested 2 to 4 weeks after the final immunization in various trials by both the HIV-SELECTEST and other HIV serodetection kits. Canarypox virus vaccine constructs used in the RV124 and HVTN 203 trials contained the p6 epitope used in the new assay. Additionally, the protein booster in the RV124 trial was gp160. In contrast, the vaccine constructs used in the VRC 004 and VRC 006 trials lacked the peptide sequences used in the HIV-SELECTEST.
Summary of HIV-SELECTEST reactivities with vaccine trial samples
The RV124 trial represents the worst-case scenario, wherein all the peptide sequences used in the HIV-SELECTEST were part of either the priming or boosting immunogens. After the last boost (day 182), 80% of vaccinees strongly seroconverted according to commercial HIV-1 detection kits, even though none were HIV infected (Table and data not shown). In contrast, only two individuals scored positive in the p6 ELISA, and none were positive in the gp41 ELISA. These findings suggest that the epitopes used in the HIV-SELECTEST were not very immunogenic in the context of the RV124 vaccine constructs.
HVTN 203 specimens included coded samples obtained from 324 trial participants prevaccination and 4 and 6 months after the first vaccination. For this panel, 30% of vaccinees seroconverted according to licensed HIV detection assays, while only 12% reacted with the p6 peptide in the HIV-SELECTEST (Table ). This finding was not surprising since the canarypox virus/HIV priming step (vCP1452) contained p6. Two specimens were also repeatedly reactive with the gp41 sequences that were not in the vaccine constructs. However, after decoding, it was confirmed that both samples were obtained from trial participants who became infected during this phase II trial.
The VRC phase I trials VRC 004, VRC 006, VRC 009, and VRC 010 were conducted in 2002-2005. The DNA plasmids (VRC 004) and nonreplicating recombinant adenovirus serotype 5 vector (rAd5) (VRC 006) used express Gag-Pol-Nef (in VRC 004) or Gag-Pol (in VRC 006) and multiclade (A, B, and C) envelope genes (gp145 in the DNA vaccine and gp140 in the rAd5 vaccine). Among the 50 participants in the VRC 004 trial, 38% (15/40) of vaccinated individuals seroconverted according to licensed HIV diagnostic kits (Table ). Unexpectedly, two samples were positive in the gp41 ELISA, one of which also reacted with p6 in the HIV-SELECTEST (Table ). Upon decoding, it was determined that the two individuals (both in the placebo arm) became infected during the VRC 004 trial (B. S. Graham et al., submitted for publication). In the VRC 006 trial (Ad5/HIV), no intercurrent HIV infections were identified, yet 60% of vaccine recipients (18/30) tested positive in licensed HIV detection tests (Graham et al., unpublished data). In contrast, none of the vaccinees reacted with either the p6 or gp41 peptides in the HIV-SELECTEST (Table ). In the VRC 009 and VRC 010 trials, subsets of DNA-vaccinated individuals (from the VRC 004 and VRC 007 trials, respectively) were boosted with the rAd5/HIV vaccine. Samples from 4 weeks postboost demonstrated a very significant increase in total HIV-specific antibodies (data not shown) and 100% seroconversion using two rapid tests (Capillus HIV-1/HIV-2 and Uni-Gold HIV tests; Trinity Biotech, NY). Importantly, all vaccinees in these trials tested negative in the HIV-SELECTEST (Table ).
Detection of intercurrent HIV infections during vaccine trials.
The data obtained with the coded panels from the HIV vaccine trials indicate that vaccine-generated antibodies are unlikely to react in the HIV-SELECTEST, especially if the vaccines do not contain the p6 sequence. Importantly, the new test detected all intercurrent infections in the blinded samples. To further determine the sensitivity of the new assay at detecting acute HIV infections in the course of vaccine trials, we tested sequential samples from HIV infections in completed phase I, phase II, and phase III trials conducted by HVTN (10
), VRC, and VaxGen (VAX 003/VAX 004 efficacy trials) (6
As shown in Table and Fig. (also data not shown), sequential samples obtained from 22 trial participants infected with HIV during the HVTN trials and the VRC 004 trial reacted positively in the HIV-SELECTEST at early time points after the estimated infection dates. Importantly, no reactivity in the HIV-SELECTEST was observed prior to HIV infection of trial participants, although they had been immunized with complex vaccine products.
HIV-SELECTEST specifically detects intercurrent HIV infections during multiple HIV vaccine trials
FIG. 2. Seroreactivities of intercurrent HIV infections during HIV vaccine trials. Reactivities in the HIV-SELECTEST of sequential plasma samples obtained from intercurrent infections in the course of the following trials are shown: (a) multiple phase I/II HIV (more ...)
Sequential samples taken soon after the first confirmed PCR-positive visit were also obtained for 65 HIV infections during the VAX 003 (AIDSVAX gp120 B/E) trial and for 81 HIV infections during the VAX 004 (AIDSVAX gp120 B/B′) trial conducted by VaxGen. The dates of PCR positivity and seroconversion by licensed HIV tests were provided by VaxGen. Table contains an analysis of two representative HIV infections in the VAX 003 and VAX 004 trials that developed strong reactivities to the p6 and gp41 peptides. Furthermore, the HIV-SELECTEST identified all intercurrent HIV infections within 90 days of PCR confirmation (Fig. for VAX 003 and VAX 004, respectively; data not shown).
Early diagnosis of intercurrent HIV infections by HIV-SELECTEST during VaxGen clinical trials VAX 003 and VAX 004a
It was also possible to compare the performance of the HIV-SELECTEST with results obtained with the FDA-licensed kits provided by VaxGen (Fig. ). In most cases, the earliest positive results were observed with the same samples for the licensed diagnostics and the HIV-SELECTEST (dots falling on the diagonal lines). Surprisingly, 24 intercurrent HIV infections in the VAX 003 trial and 25 infections in the VAX 004 trial were detected earlier with the HIV-SELECTEST than with the licensed kits (dots under the diagonal lines in Fig. ), displaying the efficacy of the HIV-SELECTEST in the early diagnosis of HIV infection. Therefore, the new assay could be part of an algorithm that will provide an important differential diagnostic tool during future phase III prophylactic vaccine trials and for testing of blood and tissue donors.
FIG. 3. Comparative reactivities of early samples from HIV-infected individuals during the VAX 003 (Thailand) and VAX 004 (United States and The Netherlands) clinical trials in the HIV-SELECTEST and licensed HIV detection kits used in the VaxGen trials. Early (more ...)