INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The need for a vaccine that is effective against human immunodeficiency virus type 1 (HIV-1) remains urgent, since the virus continues to spread worldwide (60, 155). Many candidate HIV-1 vaccines have been developed during the past decade, and several have entered clinical trials in the United States and elsewhere (14, 58). To date no efficacy trial has been conducted, and the suitability of current candidates for such trials in one or more parts of the world is a matter of vigorous debate (22, 94, 95, 106, 155). The type of vaccine that has progressed the furthest toward widespread use in humans is based on recombinant forms of the HIV-1 envelope glycoproteins (rgp120 and rgp160) (14, 58).

The first rgp120 and rgp160 immunogens were based on the sequences of HIV-1 LAI and HIV-1 SF-2, two of the earliest-isolated viruses (8, 11, 75, 133). MN rgp120 superseded LAI rgp120 in product development because the latter strain was noted to have an atypical V3 loop sequence, and at that time it was believed that V3-directed antibodies were of paramount importance for protection against HIV-1 (90). MN and SF-2 rgp120 proteins of high quality and purity, expressed in mammalian cell lines, have now been tested extensively in animals and humans; they are immunogenic and are generally regarded as safe (6, 37, 49, 56, 68, 133). Immunization with LAI Env-based immunogens has protected slightly over half of test chimpanzees from intravenous challenge with HIV-1 LAI, generally under optimal conditions (7, 8, 15, 49, 53, 143). Furthermore, both MN and SF-2 rgp120 or rgp160 have protected some chimpanzees from intravenous challenge with HIV-1 SF-2 (10, 37, 54). The significance of the chimpanzee experiments for protection of humans against HIV-1 is uncertain, principally because HIV-1 replicates only to low (LAI) or very low (SF-2) levels in chimpanzees and these strains do not cause disease in these animals. Moreover, HIV-1 LAI and HIV-1 MN are neutralization-sensitive, T-cell line-adapted (TCLA) viruses, and HIV-1 SF-2, even when grown in primary cells, is an unusual strain which is extremely sensitive to neutralization by certain reagents in vitro (101, 115, 146). These strains contrast with most primary isolates of HIV-1, which are generally relatively resistant to neutralization (98, 110, 148, 156). Furthermore, TCLA strains like LAI, MN, and SF-2 all use CXCR4 as an entry cofactor (34), whereas most strains of HIV-1 that are transmitted sexually use the CCR5 coreceptor (29, 34, 65). In contrast to the chimpanzee data, rgp120 immunogens derived from SIVmac were unable to protect rhesus macaques from intravenous challenge with the autologous, virulent virus (28). Also, immunization of cats or ponies with rgp120 immunogens derived from the feline immunodeficiency virus or the equine infectious anemia virus, respectively, failed to induce protective immunity but instead caused enhancement of disease when the animals were later challenged with virulent virus (66, 135, 151). The ambiguous and contradictory experiences with subunit vaccines in animal models therefore mandate that human trials of these immunogens be carefully analyzed, particularly with respect to individuals who become infected despite vaccination.

Two rgp120 proteins produced by Genentech, Inc., and Chiron/Biocine, Inc., based on the sequences of the clade B strains MN and SF-2, respectively, are currently under evaluation in phase I-II clinical trials conducted in the United States by the AIDS Vaccine Evaluation Group (AVEG), a contractor for the National Institutes of Health. These vaccines induce antibodies capable of neutralizing the TCLA strains of virus from which the vaccine is derived but not heterologous primary viruses (6, 9, 10, 37, 56-59, 69, 96, 98, 99, 156). The ability of these immunogens to induce cellular immunity, particularly cytotoxic T lymphocytes (CTL), is limited (5, 32, 58, 59). Partly due to these factors, efficacy trials of these products have not been carried out. Eighteen of 596 trial participants in the phase I/II trials of these proteins have become HIV-1 infected, indicating that the rgp120 vaccines gave less than complete protection from infection in the trial cohort as a whole. Here we describe analyses of the immunological responses induced in the HIV-1-infected rgp120 vaccine recipients before and after infection, we provide information on the amount of plasma HIV-1 virion-associated RNA in each infected trial participant, and we report on the env gene sequences, phenotypes, and in vitro growth characteristics of the infecting HIV-1 strains. Further information on these individuals and the clinical aspects of this study are provided elsewhere (57, 99, 127). Independent studies of two other individuals who became infected after immunization with SF-2 or MN rgp120 have been described previously (69, 100). The purpose of these studies was not to determine formally the efficacy of the rgp120 vaccines but to acquire information that could guide the design of future generations of HIV-1 vaccines.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

The Correlates of HIV-1 Immune Protection (CHIP) network. The laboratories in the CHIP consortium include those responsible for the following: confirming the infection status and monitoring the plasma viral burden (R. Connor and D. Ho), ascertaining the phenotype of the isolated viruses (E. Fenamore and R. Connor), determining the DNA sequence of the virus directly from blood (K. Kunstman and S. Wolinsky) and after isolation in vitro (F. Gao and B. Hahn), defining the humoral (A. Trkola and J. Moore) and mucosal (J. Mestecky and S. Jackson) antibody responses, determining the HIV-1-specific cell-mediated immune response (B. Walker and S. Kalams), analyzing the DNA sequences and maintaining the central database (D. McDonald, A. Neumann, and B. Korber), and evaluating epidemiological relationships (S. Vermund).

Study and control cohorts. Clinical trials of MN (Genentech Inc., South San Francisco, Calif.) and SF-2 (Chiron/Biocine Inc., Emeryville, Calif.) rgp120s are being conducted by the AVEG. By definition, individuals receiving either rgp120 immunogen or a placebo are considered vaccinees. Clinical samples were collected as described previously (56, 57, 99). During the trials, participants were monitored for HIV-1 infection by Western blot assay at 1- to 6-month intervals. Contemporary and archival serum and/or plasma samples from any individual suspected to be HIV-1 infected were sent in a blinded fashion to the central repository of the CHIP consortium at the Aaron Diamond AIDS Research Center for a variety of tests, including determination of virion-associated HIV-1 RNA levels in plasma. After the quantitative RNA data were reported to the central database at Los Alamos National Laboratory, the samples were made available for other studies in several of the consortium laboratories. Information on the specific time and use of antiretroviral therapy for the four infected vaccinees with known intercurrent treatment was available.

Clinical material. Blood obtained from the infected vaccinees at each AVEG study site was collected in tubes containing acid citrate-dextrose anticoagulant and directly shipped to the Aaron Diamond AIDS Research Center. Plasma and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque discontinuous density gradient centrifugation and used for HIV-1 isolation and for quantifying the plasma viral burden. Blood samples from the control subjects were processed by the laboratory associated with each cohort study, and the stored samples from each of the respective repositories were accessed. Parotid saliva was collected by placing Schaffer cups over Stenson's duct, as described previously (87, 134). Pre-ejaculate and ejaculate (semen), vaginal washings, and cervical, rectal, and nasal secretions were collected as described in detail in a manual for collection of human external secretions (120).

Anti-rgp120 antibody titers. The antigen capture enzyme-linked immunosorbent assay (ELISA) used has been described previously (105, 112). Briefly, ELISA plates (Immulon II; Dynatech Inc., Chantilly, Va.) wells were coated with 100 µl of a 5-µg/ml solution of sheep antibody D7324 (antibody D6205, lot D017-G; International Enzymes Inc., Fullbrook, Calif.). After the plate wells were washed, MN or SF-2 rgp120 was added at 20 or 800 ng/ml, respectively, in Tris-buffered saline (TBS) containing 10% fetal calf serum (FCS). Control wells lacked gp120. The solution concentrations of MN and SF-2 rgp120 yield an equal amount of D7324-bound gp120, as determined by the binding of CD4-immunoglobulin G (CD4-IgG) (Genentech, Inc.) (18).

ELISA for measurement of antigen-specific antibodies in mucosal samples. To determine the level of antigen-specific antibodies in mucosal samples, ELISA plates (Dynatech) were coated with MN rgp120 (Genentech, Inc.) at a concentration of 1 µg/ml overnight at 4°C. The plates were washed and blocked with 5% FCS (Mediatech, Inc., Herndon, Va.) in phosphate-buffered saline (PBS). Dilutions of external secretions were added to the plates and incubated overnight at 4°C. After being washed with PBS, the plates were developed with biotinylated F(ab)₂ fragments of goat anti-human IgG or IgA antibodies. After another washing with PBS, they were subjected to consecutive incubations with avidin-peroxidase (Sigma, St. Louis, Mo.) and 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS; Sigma). The levels of IgG and IgA anti-gp120 antibodies were measured against standard curves obtained by using solutions with known amounts of polyclonal secretory (colostral) IgA or serum IgG (Moni-Trol ES Chemistry Control; Baxter, Stone Mountain, Ga.). Serum from an individual with high levels of IgG and IgA anti-HIV-1 antibodies was used as a positive control. Negative controls consisted of external secretions and sera of noninfected individuals. The cutoff (nonspecific background binding) was set at 100 ng of IgA or IgG per ml. The levels of total IgG and IgA isotypes were determined by a capture ELISA (86, 104, 120).

Virus isolation and culture. PBMC were isolated from each vaccine recipient by standard methods as described above. The cells were serially diluted fivefold (1 × 10⁶ to approximately 3 × 10²) and cocultivated with 2 × 10⁶ phytohemagglutinin (PHA)-stimulated normal donor PBMC as previously described (17). Culture supernatants were monitored for HIV-1 p24 antigen production on days 7, 14, and 21 by a commercial enzyme immunoassay (Abbott Laboratories, North Chicago, Ill.). A culture was considered positive if the p24 value was above a cutoff value of 30 pg/ml. The virus present in the supernatant of the first positive well was propagated by a single passage (14 to 21 days) in PHA-activated PBMC and titered on PBMC to determine the 50% tissue culture infective dose (TCID₅₀). All virus stocks were aliquoted and stored at 80°C until further use. Viruses were successfully isolated from all infected vaccinees by this method. In one case (C16), it was necessary to deplete CD8⁺ T cells from the patient's PBMC prior to coculture. CD8⁺ T-cell depletion was performed with immunomagnetic beads, following the protocol of the manufacturer (Dynal, Inc.).

Phenotype determination. To assess the syncytium-inducing (SI) properties of HIV-1 strains from the vaccine recipients, MT-2 cells (5 × 10⁵) were inoculated with 100 TCID₅₀ of each isolate. The cells were washed after 24 h and monitored visually by light microscopy for the appearance of cell fusion on days 4, 7, and 10 after inoculation. Samples of the culture supernatants were also assayed for the presence of HIV-1 p24 antigen. Isolates which scored positively for both syncytia and p24 were considered SI, while those that were negative for both were considered non-SI (NSI). The growth kinetics of a subset of isolates were determined by inoculation of 100 TCID₅₀ into cultures of PHA-activated PBMC. The cells were washed after 24 h, and samples of the culture supernatants were collected and assayed for HIV-1 p24 antigen production over time.

HIV-1 coreceptor use. To assess coreceptor use by a subset of isolates from the infected vaccinees, three NSI viruses (C07, C08, and C13) and one SI virus (C20) were tested for their ability to infect HOS.CD4 cells expressing either CCR1, CCR2b, CCR3, CCR4, CCR5, or CXCR4 (30). NSI isolates from three of the putative transmitters to the infected vaccinees (pC05, pC18, and pC21) were also evaluated, as well as three NSI viruses obtained from nonvaccinated individuals during the acute phase of infection (AD60, AD74, and AD75) (12, 107). The various HOS.CD4 lines (10⁴ cells/well) were incubated with 10³ TCID₅₀ of each isolate in a final volume of 1.0 ml of Dulbecco modified Eagle medium containing 10% FCS, antibiotics, and 1.0 µg of puromycin per ml for 24 h at 37°C and then washed three times with fresh medium. Samples of the culture supernatants were assayed for HIV-1 p24 antigen on days 0, 4, 7, 10, and 14.

Virus neutralization. HIV-1 neutralization was assessed with an assay based on mitogen-stimulated PBMC as target cells and p24 antigen detection as a measure of virus output (17, 144). Briefly, serum or plasma samples from the infected vaccinees was diluted 1:8 in RPMI 1640 medium with 10% FCS, incubated with 100 TCID₅₀ of the autologous HIV-1 isolate, and added to PHA-stimulated normal donor PBMC. Following overnight incubation at 37°C, the cells were washed extensively to remove any residual serum or plasma antibodies that could interfere with the p24 antigen ELISA. Control cultures were established in duplicate by infecting cells with the autologous virus in the absence of serum or plasma. On day 7 after infection, samples of the culture supernatants were taken and assayed for HIV-1 p24 antigen. Percent neutralization was calculated by dividing the amount of p24 antigen production in the test well by the average production in the duplicate control wells. Multiple, sequential serum samples from each of the infected vaccine recipients were tested against an autologous isolate obtained at the earliest time point after HIV-1 infection had occurred.

MAbs. Monoclonal antibodies (MAbs) 2G12 and 2F5 were donated by H. Katinger (Polymun Scientific Inc., Vienna, Austria) (22, 23, 108, 116, 132, 133, 145), MAb IgG1b12 was provided by D. Burton (Scripps Research Institute, San Diego, Calif.) (16), and the CD4-IgG2 molecule was a gift from P. Maddon (Progenics Pharmaceuticals Inc., Tarrytown, N.Y.) (1). MAb 447/52-D was purchased from Cellular Products Inc. (Buffalo, N.Y.) (55). Murine MAb B13 was a gift from G. Lewis (Institute of Human Virology, Baltimore, Md.) (111).

Cell lines. Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCL) were established from PBMC obtained from each of the 18 individuals, as described previously (149). The transformed B-LCL were maintained in RPMI 1640 medium containing 20% FCS supplemented with 2 mM L-glutamine, 50 U of penicillin per ml, and 10 mM HEPES.

Limiting-dilution assays of CTL precursors. Limiting-dilution precursor frequency analysis was used to determine the relative strength of the postinfection CTL response against HIV-1 proteins, as described previously (83, 84). PBMC were cultured at 250 to 16,000 cells per well in 24 replicate wells of a 96-well microtiter plate. Gamma-irradiated (30 Gy) PBMC (2.5 × 10⁴) from an HIV-1-seronegative donor were added to each well with 0.1 mg of the anti-CD3 MAb 12F6 per ml. Ten to 14 days later, the cells were split and assayed for cytotoxicity on ⁵¹Cr-labeled autologous B-LCL infected with recombinant vaccinia viruses expressing HIV-1 Gag (vABT141) (Therion Biologics, Cambridge, Mass.), reverse transcriptase (vCF21), Nef, or Env proteins. The Env proteins were derived from the LAI (vPE11 and vABT299), MN (vMN462), RF (vRF222), or SF2 (Vpe11) strain (74, 84, 142, 149). The fraction of nonresponding wells was determined as the number of wells in which the ⁵¹Cr released did not exceed the mean plus three standard deviations of the spontaneously released ⁵¹Cr of the 24 control wells divided by the number of assayed wells. Activated-cell frequency was estimated by the maximum-likelihood method (19, 31).

HIV-1 RNA assay. HIV-1 virion-associated RNA in the serum or plasma of infected vaccine recipients was measured by the Amplicor HIV-1 Monitor assay (Roche Molecular Systems). Briefly, total RNA was extracted from 200 µl of either serum or plasma collected in acid citrate-dextrose to which a standard, HIV-1-unrelated RNA preparation of known copy number was added. A 142-bp sequence of the HIV-1 gag gene was reverse transcribed and, in the same reaction, amplified by PCR for 30 cycles. The amplified products were serially diluted fivefold and hybridized to oligonucleotide probes coated on the wells of a microtiter plate. Following hybridization, the plates were washed extensively and the bound products were detected with an avidin-horseradish peroxidase conjugate. The HIV-1 RNA copy number was calculated on the basis of the ratio of the absorbance (OD₄₅₀) reading from the bound HIV-1 products relative to that from the internal-standard RNA. The results are expressed as HIV-1 RNA copies per milliliter of serum or plasma. The results of HIV-1 RNA quantitation for samples from the nonvaccinated control subjects did not show a log-normal distribution, possibly due to the differences in sample collection procedures between the different cohorts. Therefore, nonparametric statistics were used to compare the RNA values of the infected vaccinee cases to those of the controls.

DNA sequencing. env genes were amplified directly from uncultured PBMC DNA (gp120) and from cultured PBMC DNA (gp160) from each individual and then cloned and sequenced as described previously (153). The gp120 sequences from PBMC DNA taken directly from the study subject were determined at Northwestern University by methods described previously (153, 154), and the gp160 sequences from primary-culture DNA were determined at the University of Alabama as described elsewhere (51). The use of these two methods, in two different laboratories, ensured the integrity of both the PCR products and the viral isolates (81, 91).

Viral sequence analysis. Comparisons were based on various breakdowns of the available PCR product sequence data sets. Initial sequence alignments were generated by using a hidden Markov model (HMMER, version 1.8; http://genome.wustl.edu/eddy/HMMER/main.html) (36, 117). Both DNA and protein alignments were hand edited by using the multiple alignment sequence editor (39). Simple sequence similarity comparisons were made by using the multiple alignment sequence editor after removing positions in the alignment at which gaps had been inserted to maintain the alignment. These were calculated as Hamming distances, or (1  s) × 100, where s is the fraction of shared sites in two aligned nucleotide sequences (80).

Nucleotide sequence accession numbers. Viral sequences generated for this study have been submitted to GenBank under accession no. U84792 through U84887.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Immunization and infection profiles. The 401 study group comprised all individuals who became infected during phase I-II trials of subunit or composite vaccines conducted by the AVEG. Enrollment in the 401 study began in January 1994 and ended in June 1995. In this paper, we report exclusively on 18 individuals who participated in trials of rgp120 (MN or SF-2) and yet became infected with HIV-1 despite intensive counseling to avoid high-risk behaviors (57, 60, 127). Several other recipients of different immunogens have become HIV-1 infected, and they are described elsewhere (57, 69, 100).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Temporal relationship between entry into the vaccine trial, receipt of immunizations, and the period during which HIV-1 infection occurred. The date of entry (first immunization) into one of the vaccine trials is designated as day 0, and all time points represent days elapsed from that date. Arrows indicate days on which immunization occurred, and hatched bars correspond to the interval between the last negative and first positive HIV-1 RNA PCR result, based on measurement of viral RNA in plasma or serum. The cases are arranged in order of the time between commencement of immunization and the time of HIV-1 infection.

View larger version (171K): [in this window] [in a new window]   FIG. 2.   Longitudinal profiles of plasma viral burden, and antibody responses to MN and SF-2 rgp120, for the 18 infected vaccinees. Each panel represents one of the 18 infected vaccinees; the corresponding case number and respective immunogen are shown in the upper left-hand corner of each profile. The red triangles represent the level of plasma HIV-1 virion-associated RNA in copies per milliliter, the blue circles represent the midpoint antibody titers to SF-2 rgp120, and the yellow squares represent midpoint antibody titers to MN rgp120. Day 0 designates the date of entry into one of the vaccine trials, and all time points represent days elapsed from that point. The days on which immunizations were received are indicated by arrows. Four individuals (C04, C15, C20, and C26) received antiretroviral therapy following HIV-1 infection while still participating in a vaccine trial. The day(s) on which therapy was initiated is indicated by an asterisk. For two cases (C15 and C26), treatment was initiated after the last time point in which viral RNA levels and antibody responses were tested. C04 was treated with dideoxyinosine (beginning day 498) and AZT (beginning on day 658); C15 was treated with dideoxycytosine (beginning on day 490); C20 was treated with AZT (beginning on day 811); and C26 was treated with AZT, 3TC, and Retonavir (beginning on day 976).

Antibody responses. (i) gp120 binding antibodies. To gain an understanding of the immune response of each individual to MN or SF-2 rgp120 proteins, we measured serum anti-gp120 IgG midpoint titers to both proteins by using an antigen capture ELISA. Every serum sample was titrated against both rgp120s under conditions in which a control HIV-1-positive serum specimen and the CD4-IgG molecule bound equivalently to each rgp120 (data not shown). Thus, antibody titers to the two rgp120 proteins may be compared within and between each longitudinal profile (Fig. 2). Considering only those 11 individuals who received three or more immunizations with rgp120 before becoming infected, several general observations may be made.

(ii) Neutralizing antibodies. Titers of antibodies to monomeric gp120 are not predictive of primary-virus neutralization titers (48, 108, 110, 147, 148). We therefore tested whether serum taken from each vaccinee before HIV-1 infection could neutralize the HIV-1 strain isolated from the same individual soon after infection. The isolated viruses were cultured only in mitogen-stimulated PBMC (see below) to avoid selection of neutralization-sensitive strains. Neutralization of these primary viruses was assessed in a well-characterized assay that uses mitogen-stimulated PBMC as target cells and p24 antigen output as a measure of virus replication (17, 144). Vaccinee sera were tested at a 1:8 dilution, since higher concentrations may lead to nonspecific inhibition of cell growth or virus replication. At this dilution, no serum sample from any vaccinee at any time point prior to infection was able to reduce the infectivity of the autologous isolate by 90% (data not shown). A few sera from some individuals sporadically caused 50% neutralization, but the significance of a twofold reduction in HIV-1 infectivity is questionable.

Mucosal antibodies. The levels of antigen-specific and total antibodies in external secretions, including parotid saliva, pre-ejaculate, and seminal plasma, and in secretions collected by vaginal washing, uterine cervical wicking, nasal washing, and rectal wicking were determined. Results of ELISA analyses of secretions from nine infected vaccinees, two placebo recipients, and two putative transmitters are presented in Table 4. Antibodies of the IgG isotype specific for MN rgp120 were detected in the genital tract secretions of three infected vaccinees (C05, C09, and C10) and one putative transmitter (pC12). IgG antibodies were also detected in seminal fluid from three males and a vaginal wash and cervical wick from one female. One male vaccinee positive for IgG antibodies in seminal plasma (C10) had IgA anti-MN rgp120 antibodies in his parotid saliva as detected by ELISA. The isotype association of HIV-1-specific antibodies in these secretions reflected the predominant isotype present in the respective secretions. Thus, total immunoglobulins in both male and female genital tract secretions were predominantly of the IgG isotype. External secretions from the remaining infected vaccinees did not contain antigen-specific antibodies at levels above the cutoff (100 ng/ml). When sufficient volumes of external secretions were available, the samples were also analyzed by Western blotting to confirm the results obtained by ELISA and to determine the specificity of antibodies (Table 5). IgG antibodies to gp120 were detected in one sample of seminal plasma (C09), in one sample from a putative transmitter (pC12), and in cervicovaginal secretions from one female (C05). Parotid saliva from C10 contained IgG, but not IgA, antibodies to gp120, gp41, and p24. With the exception of C10 (for whom IgA antibodies were detected in parotid saliva by ELISA but not by Western blotting), concordant results were obtained. These data indicate that systemic rgp120 immunization of these volunteers was ineffective at inducing mucosal immune responses manifested by the presence of secretory IgA antibodies.

Limiting-dilution analysis of CTL precursors. CTL precursor frequency analysis was performed to determine whether vaccination with soluble rgp120 might have skewed the postinfection response against envelope antigens. Table 6 shows the results for nine of the individuals from whom B-LCL were established. Background precursor frequencies against the control vaccinia virus (NYCBH) were generally low for each individual (<50 of 10⁶ cells). A result was considered significant if >50 of 10⁶ cells (corresponding to a precursor frequency of 1 in 20,000) were scored positive with a background level of <50 per 10⁶ cells. Alternatively, if the background was between 50 and 100 per 10⁶ cells, a result of >150 per 10⁶ cells was considered significant. Five of nine subjects (C06, C10, C11, C13, and C21) showed Gag-specific CTL responses for at least one of the time points studied. Two subjects (C06 and C20) generated reverse transcriptase-specific CTL precursors, and two (C06 and C21) had Nef-specific precursors. Env-specific CTL responses were infrequent and were not vigorous. The highest Env-specific response was detected in subject C06, but this was specific for the LAI envelope and not the MN strain with which this individual had been immunized. In fact, none of the rgp120-vaccinated individuals had detectable Env-specific CTL responses to the immunizing strain of HIV-1. One placebo recipient (C18) was, ironically, the only individual with a postinfection cellular immune response to the MN strain. There were no SF2-specific CTL responses.

Phenotypes and growth characteristics of infecting HIV-1 strains. HIV-1 strains were isolated in PBMC cocultures from the 18 infected vaccinees and from 6 of the putative transmitters. For only one subject (C16) was depletion of CD8⁺ T cells prior to culture required to obtain a virus isolate, indicating that virus replication in vitro was sufficient to overcome CD8⁺-T-cell-mediated suppressive effects in the majority of cases. All 24 viruses grew well in mitogen-stimulated PBMC; representative growth rates for several strains are shown in Fig. 3. The in vitro replication rates of viruses isolated from the infected vaccine recipients were not significantly different from those of isolates from the putative transmitters and from nonvaccinated, acutely infected individuals (Fig. 3 and data not shown). It is common for HIV-1 strains isolated soon after primary infection to replicate relatively slowly in culture (26, 45, 46). Our results indicate that it is unlikely that prior immunization with rgp120 selected for the transmission of only poorly replicating HIV-1 strains to the infected vaccinees.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Replication kinetics of viral isolates from infected vaccinees in activated PBMC. The replication kinetics of selected HIV-1 isolates from the infected vaccinees were evaluated in cultures of PHA-stimulated normal donor PBMC. Cells were infected with 100 TCID50 of each isolate, and the levels of HIV-1 p24 antigen were measured in culture supernatants on days 0, 3, 7, 10, and 14 after inoculation. Isolate pC13 is from the partner of C13; isolates AD6 and AD13 were obtained from nonvaccinated acute seroconvertors.

View larger version (49K): [in this window] [in a new window]   FIG. 4.   The antigenic domain in the V3 loop. All available sequences are included, grouped by individual, and aligned with the MN consensus sequence. An example of a matrix for a Fisher's exact test, used to examine the possibility of greater diversity in the MN recipient strains relative to MN compared to the control (CONT) set, is shown at the bottom. The number of amino acid changes relative to the vaccine strain sequence for the closest sequence in each of the vaccinees and for each of the controls was tallied (138, 139). For example, C06 had three changes relative to MN in this region. The number of individuals with viruses carrying a given number of mutations was tallied for the MN vaccine recipient group and for the controls, and the two groups were not significantly different by this measure. The positions that tend to correlate with the SI phenotype are the first and last amino acids of this region, and the two SI isolates from among the vaccine recipients (C04 and C20) carry positively charged arginine and lysine residues in these positions, respectively. Dashes indicate gaps in the sequence alignments.

Coreceptor use by the infecting HIV-1 strains. Macrophage-tropic, NSI isolates of HIV-1 preferentially use CCR5 as a coreceptor for infection (26, 33, 136) and are phenotypically characteristic of the majority of HIV-1 strains isolated during primary infection (158). We assessed the coreceptor requirements of three NSI isolates (C07, C08, and C13) and one SI isolate (C20) from the infected vaccinees, along with three NSI isolates from putative transmittors (pC05, pC18, and pC21) and three additional NSI isolates from nonvaccinated individuals, obtained during primary HIV-1 infection (AD60, AD74, and AD75). We found that NSI isolates from the infected vaccinees, donors, and nonvaccinated controls all used exclusively CCR5 for infection, while the SI isolate used a broader range of coreceptors, including CCR5, CCR2b, CCR3, and CXCR4 (data not shown). Although the number of isolates tested was too small to draw formal conclusions, these results suggest that prior immunization with rgp120 does not result in the selection of phenotypic variants with distinct coreceptor requirements. Rather, the isolates from the infected vaccinees displayed phenotypic profiles and coreceptor usage patterns consistent with most sexually transmitted strains of HIV-1 (29, 65).

Virus burdens in infected vaccine recipients. Irrespective of any effect of the immunization regimen, the 18 individuals we examined became infected with HIV-1. The most direct evidence for this was the detection of HIV-1 virion-associated RNA in the plasma of each individual by a sensitive and quantitative PCR-based assay (72, 102). The levels of plasma HIV-1 virion-associated RNA for each of the participants, superimposed on their antibody responses to the immunogens, are shown in Fig. 2. Although there was considerable variation between individuals, in general we observed a sharp increase in the viral burden soon after infection followed by a decline over time. However, because of infrequent sampling intervals, we had no way of determining the true peak viral burdens, thus preventing analysis of this parameter. In all cases, the HIV-1 RNA levels in the plasma reached an approximate steady state, ranging from 750 to 48,000 copies/ml at 9 to 12 months after infection.

View larger version (12K): [in this window] [in a new window]   FIG. 5.   Comparison of the viral loads of infected vaccinated individuals and matched controls at 9 to 12 months after the estimated time of infection. Viral RNA copy numbers in sera or plasma from HIV-1 infected individuals who had been given an SF2 rgp120 vaccine are labeled in blue, and those who had received MN rgp120 are in green. Individuals who had received three or four vaccinations prior to infection are indicated by closed circles; those who had less than three vaccinations are indicated by open circles. The single individual who had documented antiretroviral agent (AZT) use prior to this sampling period is indicated with an arrow. In cases for which two samples from a single individual were available during this 3-month period, the average value was used. Twenty-three controls matched for risk factors for infection, gender, age (±10 years), and year of seroconversion were obtained from four different cohorts, excluding samples that were derived from patients with prior use of antiretroviral agents and patients for whom no samples were available in the appropriate time frame of 9 to 12 months postseroconversion. Three samples from individuals given placebo vaccinations prior to infection were included with the controls and are marked as open circles in the first column of controls. The red circles are for placebo recipients from the 401 study, and the pink circle is a placebo recipient from another U.S. vaccine trial. The other four columns display control RNA values from the four different cohorts: the TRUNK cohort (), the ALIVE cohort (), the MACS cohort (×), and HIVNET (). The medians and interquartile ranges for the complete control set and the complete vaccine recipient set are indicated by the thick and thin horizontal black bars, respectively.

Viral sequence analysis. DNA sequences of the gp120 and gp160 regions of env from uncultured and cultured PBMCs, respectively, from each of the infected vaccinees and their matched controls (see above) were obtained. These are presented as deduced amino acid sequences in Fig. 6. Between one and five sequences were generated for each of the vaccine recipients, and a single representative sequence was generated for each of the matched controls. Whenever possible, vaccinee sequences were generated by using two different strategies in parallel in two different laboratories. A sequence from each vaccinee was generated from cultured PBMC. Cloning and sequencing directly from the study subjects' PBMC were performed when there was a target copy number adequate for direct amplification by PCR (i.e., in 15 of 18 cases).

View larger version (109K): [in this window] [in a new window]   View larger version (113K): [in this window] [in a new window]   FIG. 6.   Amino acid alignment of gp120 protein sequences. Only a single sequence is shown per individual vaccine recipient, although multiple sequences were determined. Yellow boxes indicate antigenic regions in V2, V3, and C4 that were analyzed in detail. Gray shading indicates distinctive amino acid signature sites. Purple boxes indicate the most distinctive motifs identified with MotifScan. All sequences are aligned to the consensus sequence of the control set, labeled CON. The sequence designations include the study subject identification number, the clone number, and either an H (if sequenced at the University of Alabama at Birmingham) or a W (if sequenced at Northwestern University). Dashes indicate identity with the B subtype sequence at the top of the alignment; periods indicate insertions made to maintain the alignment; pound signs indicate frameshift mutations.

Phylogenetic analyses. Phylogenetic reconstructions of the relationships between viral sequences from the infected vaccinees and those from the matched controls confirmed the authenticity of the samples: in neighbor-joining trees, each of the within-subject sequence sets formed a distinctive clade preserved in 100 of 100 resamplings in bootstrap analysis (Fig. 7). Three of the four epidemiologically linked pairs, all except vaccine recipient C13 and his partner pC13, were closely associated in phylogenetic analysis, again preserved in 100 of 100 bootstrap tests. A comparison with matched case controls (Fig. 7) and a number of contemporary and archival viral sequences (data not shown) showed that all vaccinee and control sequences in this study were from the B clade and that they formed an unstructured topological pattern, with one notable exception. Sequences from four individuals (two infected vaccine recipient cases, C17 and C24, and two control cases, V21 and V25) were associated both in the neighbor-joining tree shown in Fig. 7, through bootstrap analysis (in 79 of 100 bootstrap tests), and in a maximum-likelihood phylogenetic reconstruction (data not shown). Retrospective examination of clinical records, undertaken by special request because of the observed clustering pattern, revealed that the four study subjects with phylogenetically linked viral sequences each had intravenous drug use as their primary risk factor for infection, and all four attended the same clinic in the same city.

View larger version (33K): [in this window] [in a new window]   FIG. 7.   Vaccine breakthrough sequences and controls: neighbor-joining phylogenetic tree, with bootstrap values, showing the relationships between the viral sequences from vaccinated individuals and matched controls. Partne