Sexual Transmission of Single Human Immunodeficien
http://www.100md.com
病菌学杂志 2005年第22期
The Peter Medawar Building for Pathogen Research and The James Martin 21st Century School, Nuffield Department of Clinical Medicine, University of Oxford, South Parks Road, Oxford OX1 3SY, United Kingdom
Jefferiss Research Laboratories, Wright-Fleming Institute, Imperial College London, St. Mary's Hospital, Norfolk Place, London W2 1PG, United Kingdom
Department of Immunohaematology and Blood Transfusion, Leiden University Medical Centre, P.O. Box 9600, 2300 Leiden, The Netherlands
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, United Kingdom
ABSTRACT
Antigenic variation inherent in human immunodeficiency virus type 1 (HIV-1) virions that successfully instigate new infections transferred by sex has not been well defined. Yet this is the viral "challenge" which any vaccine-induced immunity must deal with. Closely timed comparisons of the virus circulating in the "donor" and that which initiates new infection are difficult to carry out rigorously, as suitable samples are very hard to get in the face of ethical hurdles. Here we investigate HIV-1 variation in four homosexual couples where we sampled blood from both parties within several weeks of the estimated transmission event. We analyzed variation within highly immunogenic HIV-1 internal proteins encoding epitopes recognized by cytotoxic Tlymphocytes (CTLs). These responses are believed to be crucial as a means of containing viral replication. In the donors we detected virions capable of evading host CTL recognition at several linked epitopes of distinct HLA class I restriction. When a donor transmitted escape variants to a recipient with whom he had HLA class I molecules in common, the recipient's CTL response to those epitopes was prevented, thus impeding adequate viral control. In addition, we show that even when HLA class I alleles are disparate in the transmitting couple, a single polymorphism can abolish CTL recognition of an overlapping epitope of distinct restriction and so confer immune escape properties to the recipient's seroconversion virus. In donors who are themselves controlling an early, acute infection, the precise timing of onward transmission is a crucial determinant of the viral variants available to compose the inoculum.
INTRODUCTION
A thoroughdescription of the human immunodeficiency virus type I (HIV-1) inoculum that establishes new infection is central to understanding protective immunity to this virus. Close sequence similarity in envelope has been observed in HIV-1 successfully transmitted between heterosexual African partners (12). In contrast, viral strains resistant to neutralization by antibody are infrequently transmitted (12). However, HLA class I-restricted antigenic properties of the successful infecting virus are largely unknown. Useful comparisons between "donor" HIV-1 variants and the virions that establish infection in a new host require sampling very close to the transmission event.
Here we investigate the extent to which the founding HIV-1 population represents the antigenic properties of the virus in the donor. Much evidence now supports the concept that HLA class I-restricted CD8+ T-cell responses are crucial for the control of HIV and simian immunodeficiency virus infection (8, 20, 23, 30, 38). This evidence, which implies that cytotoxic T lymphocytes (CTLs) can inhibit retroviral replication, has been used to justify vaccines that rely solely on the induction of this form of immunity for protection of na?ve subjects (6). However, the CTL response to HIV-1 selects for escape variants (9, 18, 32, 33), which can impair completely the usefulness of such vaccines (3, 4).
The transmission of CTL escape variants from mother to child has been well documented (16, 17) because transmitter pairs are readily identified. Evidence has been adduced that CTL escape variants can be transmitted sexually (2, 15, 16, 27). To make this inference, an association in chronically infected patients between the possession of a particular HLA class I allele and the presence of an escape variant was first established. When the same variant was found in recently infected patients that did not have the HLA class I molecule required for a response against the epitope, it was concluded, on reasonable grounds, that the polymorphism had been transmitted.
In the present study we set out to compare, as closely as possible, the extent to which individual virions could transfer genetically linked immune escape properties. We reasoned that immune escape was positively selected during HIV infection. Given the enormous extent of recombination in the virus, further reassortment and selection could lead to the outgrowth of single virions bearing several epitopes with immune escape properties. Once we had identified such viruses, we asked whether this "multi-immune-escape HIV" could be transmitted. If the new host shared HLA class I with the donor, then it is very possible that the recipient’s control of the virus could be undermined.
We examined the antigenic properties of HIV-1 either side of transmission in four homosexual transmitter pairs. By focusing on the immunogenic internal viral proteins, we demonstrate that CTL escape variants are readily transmitted, so that infection can be established by a virus able to simultaneously evade multiple CTL responses.
MATERIALS AND METHODS
Patient pairs. Patients attending the HIV clinic at St. Mary's Hospital, London, United Kingdom, with acute HIV infection were recruited to a study (SPARTAC) of a short-course highly active retroviral therapy (HAART). As a substudy, consenting patients were asked to identify recent sexual partners. Transmitting couples were only studied if both the recipient and the donor gave written consent.
This study was granted local research ethical approval to recruit both newly infected patients and their HIV-transmitting partners. Acute HIV infection was identified according to the following definition: previously documented HIV antibody negative within 6 months, negative antibody test with positive HIV RNA PCR, or incident infection using the Abbott detuned assay. Detailed sexual histories were taken from both partners separately.
Viral sequencing and analysis. Plasma samples containing the virus were obtained from both donor and recipient within 2 to 6 weeks of the time when acute infection was diagnosed in the recipient. HIV-1 virion RNA was reverse transcribed, amplified, cloned, and sequenced from several time points close to the time of transmission, according to a previously described protocol (31). The clonal sequencing effort was directed toward, but not limited to, the highly immunogenic gag and nef regions (1). As a precaution against contamination, all reagents were aliquoted before use, PCR amplification of the donor and recipient of each pair was spatially and temporally separated in the laboratory, and negative controls were conducted in parallel at each stage of PCR.
Phylogenetic comparison of the four transmitter pairs. Nucleotide pol sequences (1,269 bp), obtained by population sequencing and incorporating the entire protease gene and a part of the reverse transcriptase (RT) gene, were manually aligned together using the program McClade version 4.07 (28). Sequences from the four transmitter pairs; HIV-1 clade A, B, C, and D consensus sequences; and 78 pol RT sequences from acutely HIV-infected individuals attending St. Mary's Hospital, London, United Kingdom, during the period 2001 to 2004 were incorporated into the alignment. The matrix was subsequently imported into the tree building software PAUP (39), and an initial neighbor-joining (NJ) tree (36) was reconstructed under the Hasegawa-Kishino-Yano (HKY85) model of evolution (19). The NJ topology was used as a starting tree for a heuristic search for a maximum-likelihood tree (13) under the general time reversible model of nucleotide substitution (42), with proportion of invariable sites and rate heterogeneity. The parametric values of the selected model are available from the authors on request. The robustness of the NJ phylogeny was assessed by bootstrap analysis (14), with 1,000 rounds of replication.
Gamma interferon ELISPOT assay. Antigen-specific responses from patients' peripheral blood mononuclear cells (PBMCs) were measured using synthetic peptides (Invitrogen, Mimotopes, Biosynthesis) and a standard enzyme-linked immunospot (ELISPOT) assay, as previously described (24). All assays were done in duplicate. A duplicate negative control was included in all assays; the number of spots in the negative control was subtracted, and the results were normalized to give the number of spot-forming cells per million PBMCs.
Peptide binding assay. The competitive fluorescence binding assay was carried out as described previously (21). In brief, B-cell lines bearing the relevant HLA class I molecules were subjected to acid elution for 90 s at pH 3.1 (for HLA-A03-restricted peptides, a pH of 2.9 was used). The B cells were then incubated for 24 h at 2 to 8°C with an HLA-specific reference peptide conjugated to a fluorescein label and a test peptide titrated between 184 μM and 0.02 μM. After the incubation, each sample was stained with 7-aminoactinomycin D (Viaprobe; BD Biosciences) in order to exclude dead cells from subsequent analysis, fixed, and analyzed by flow cytometry. All assays were done three times. The inhibition of fluorescein-labeled reference peptide binding, through competition with the test peptide, is a measure of the binding affinity of the test peptides. Inhibition was determined as previously described (21) and expressed as the concentration that results in a 50% reduction in fluorescence (IC50). The percent reduction in binding affinity (%RBA) of a variant peptide (V), relative to the consensus (index, I) was calculated using the following formula: %RBA = [1 – (IC50I/IC50V)] x 100.
Defining immune escape variants. A polymorphism within an HLA class I-restricted epitope was defined as conferring escape if it satisfied one or more of the following criteria: (i) the epitope variant could not bind to the relevant class I allele (and hence cannot be presented to CTL), (ii) the variant was not recognized by the host (autologous) CTL although the consensus epitope was recognized, or (iii) the variant was not recognized by three sets of heterologous PBMCs. Binding assays were performed on around 90% of the analyzed variants. Wherever possible, the variants that bound to HLA were also tested for immunogenicity on autologous PBMCs by ELISPOT. The heterologous PBMCs, which can respond to the consensus epitope, were taken from HIV-infected patients and were used only when the autologous PBMCs were not available.
In the assay used for the evaluation of binding, a reduction of >99% represents a case when, at the highest concentration of the test peptide (200 μM), there was no displacement of the fluorescently labeled index peptide. In our definition of escape through lack of binding, only peptides for which binding was reduced >99% were taken as escape variants. For peptides that showed reduced (but not completely abolished) binding, immunogenic responses were also tested in order to establish the escape status of the peptide. Failure of recognition by autologous or heterologous PBMCs was recorded when there was no gamma interferon production over background (as tested by ELISPOT) at peptide concentrations of 1 μM or lower.
RESULTS
Four male homosexual patient couples were identified after diagnosis of acute HIV infection in the recipient. A pair is referred to as donor (Dn) and recipient (Rn), where n is the pair number. The times when patients were recruited and sampled are shown in Fig. 1a to d by black arrows. Orange arrows mark the time when samples were analyzed to establish the relatedness of viral sequences closest to the time of transmission. The periods of HAART are denoted by dark green bars (Fig. 1).
The close relatedness of the viruses harbored within transmission pairs was determined unambiguously by the topology of the maximum-likelihood tree (Fig. 2). For each couple, the pol RT sequences from the donor and recipient clustered tightly together. On testing the tree topology for robustness, the transmitter clusters occurred repeatedly (100% bootstrap support). The genetic distances were consistently shorter between sequences involved in transmission pairs than between unlinked sequences (average within-pair genetic distance of 0.0024 substitution/site), and transmission pairs 1 and 3 had identical sequences. All pairs, except pair 1, were infected with an HIV-1 clade B virus. Other putative transmission networks were also observed within the phylogeny, although these were not analyzed in the present study.
Clonal sequencing of the viruses present in the donors and the recipients was carried out. Consensus sequences corresponding to relevant clades were taken from the National Institutes of Health/National Institute of Allergy and Infectious Diseases databases (http://www.hiv.lanl.gov). In total, 61 epitopes were sequenced among the four transmitter pairs. Of these, 47 epitope sites showed the presence of variants. Variant and consensus peptides were synthesized and immunologically characterized by binding assays or CTL recognition or both. Variants were then scored for "escape" according to the strict definition described in the Materials and Methods section. Table 1 shows the number of epitopes in each transmitter couple that had variants that were defined as escape by virtue of not binding or lack of ELISPOT response by autologous or heterologous PBMCs. The binding characteristics of 38 of these variants and consensus peptides are reported in Table 2.
Detection of selection of escape in HIV transmitters: sample timing is crucial. D1 was recruited with primary HIV infection. He was given a short course of HAART and then experienced viral rebound (Fig. 1a). Recipient R1, who was infected by D1 (Fig. 2) almost certainly during that rebound, was sampled at the nominal time week 0 (Fig. 1a). This allowed very close HIV sequence comparisons in four samples taken from D1 before and after week 0 (Fig. 1a, D1, green inverted triangles). We found that the sequence within the p17 Gag HLA A0301-restricted epitope RLRPGGKKK in R1 (RLRPGGRKK; substitutions are underlined throughout) was identical to that detected in D1 19 weeks before R1 was first sampled, i.e., week 0 (Fig. 3). Variant RLRPGGRKT, which was not recognized by D1 and had 95% reduction in binding affinity, subsequently rose to fixation in D1. This variant was shown to be under positive selection in the donor (using the program Codeml from the PAML software package) (43). We did not find this escape variant in R1, but this is not surprising since this sequence rose from 19% to fixation in D1 20 weeks after R1 was recruited (Fig. 3, week 20). This example illustrates that the precise moment during the natural course of HIV infection at which transmission takes place can be a critical determinant as to which viral variant infects the new host (Fig. 4 and 5). This is particularly true during acute infection when the successful inoculum is rapidly adapting to a new host. Escape variants were transmitted from D1 to R1 at four other epitopes (Table 1).
Sexual transmission of HIV-1 bearing multisite CTL escape variants. We identified CTL escape variants in three of the donors (D1, D2, and D3). Some of these escape variants were transmitted (Table 1). In pair 1, 4 of 5 escape variants were transmitted; in pair 2, 8 of 10. Out of a total of 21 epitopes that were immunologically characterized, 16 encoded escape variants in the donor virus population very close (i.e., 1 to 11 weeks) to the estimated time of transmission. CTL escape variants at 12 of these sites were transmitted and detected in samples taken from the recipients when they were first identified with acute HIV infection (week 0).
Of particular interest is that viral segments bearing several linked CTL escape epitopes were identified in two donors (D1 and D2). In D1, who was infected with HIV subtype A, escape variants were present at high frequency in three epitopes of distinct HLA class I restriction within Gag p17 at 4 and 19 weeks before R1 was first recruited (Fig. 4). In D1, a single change of K to R (K26R) at the C-terminal end of the HLA-A0301-restricted KIRLRPGGK epitope led to a loss of recognition (Fig. 4, A3 inset) and a 74% reduction in HLA binding affinity (Table 2, KIRLRPGGR). For the HLA-B0801-restricted GGKKKYRL (GL8) epitope, the K26R change and R30K change (GGRKKYKL) resulted in failure of recognition by HLA-B0801-restricted T cells specific for the GL8 epitope (Fig. 4, B8 inset). An additional instance of escape in D1 was observed in the HLA-A0101-restricted GTEELRSLY epitope: the GTEELRSLF variant found in D1 was not recognized by heterologous PBMCs at low peptide concentrations (Fig. 4, A1 inset). Binding assays showed that the change of Y to F (Y79F) had reduced the HLA-A0101 binding affinity of the peptide by 98% (Table 2).
Therefore, in D1 we detected single virions bearing polymorphisms at three epitopes of distinct HLA class I restriction, all of which individually conferred immune escape. Virions containing the identical three linked escape variants were also found in all of the sequenced clones in R1 during acute HIV infection (week 0) and were propagated for 52 weeks (Fig. 4). According to a detailed clinical history of each patient, transmission from D1 to R1 is most likely to have occurred 3 to 6 weeks prior to week 0. There was no evidence of reversion of these polymorphisms in R1 during this period.
Transmission of escape variants abolished CTL recognition in the recipient who shared HLA alleles with his donor. For donor-recipient pair 2, who share HLA-A0101 and HLA-B0801, transmission is likely to have occurred 4 to 9 weeks prior to week 0 (the time R2 was first sampled) (Fig. 5). Variants were detected at four epitopes in D2 on a continuous sequence encoding Gag p17, collected from samples 11 and 22 weeks before R2 was recruited (RLRPGGKKQ, GGK KQYRL, GSEEIKSLY, and DVKGTKEAL). These variants were not recognized by CTL in ELISPOT assays (Fig. 5, insets).
A change of K to Q (K28Q) at the C-terminal end of the HLA-A0301-restricted RLRPGGKKK epitope was not recognized, and the variant RLRPGGKKQ had a reduced binding affinity of >99% (Table 2). As well as directly leading to escape in the RLRPGGKKK epitope, this change has been shown to confer escape in the neighboring HLA-A3-restricted KIRLRPGGK epitope by interfering with its processing (2).
The K28Q and the K30R change of the HLA-B0801-restricted overlapping GGKKKYKL epitope are associated with a loss of recognition. The K28Q polymorphism lies at an anchor residue in HLA-B0801-restricted epitopes, and the K30R change can lead to antagonism of the GGKKKYKL response (35). The GGKKQYRL variant peptide sequence, detected in both D2 and R2, had an 82% reduction in binding affinity assays (Table 2).
Variants at the HLA-A0101-restricted GSEELRSLY (variant GSEEIKSLY) and HLA-B0801-restricted EVK DTKEAL (variant DVKGTKEAL) epitopes were also tested in ELISPOT assays. The GSEEIKSLY variant showed a 44% reduction in binding affinity (Table 2). Neither variant was recognized in ELISPOT assays (Fig. 5, A1 and B8 insets). The escape variant DVKGTKEAL was not transmitted. As in D1, individual virions encoding immune escape variants at several linked sites were found in Gag p17 in D2. With the exception of the EVKDTKEAL epitope, all Gag p17 substitutions responsible for escape were present at high frequency in R2.
Escape variants were transmitted from D2 to R2 at an additional two sites, HLA-B0801-restricted YLQDQQLL in Env gp41 and HLA-A0101-restricted ISERILSTF in Rev. Both of these variants had over 90% reduction in binding affinity (Table 2).
Despite the absence of a detectable response to escape variants encoded at four epitopes, R2 mounted a response to the HLA-B0801-restricted FLKEKGGL Nef epitope, and the HLA-B0801-restricted variants DVKDTKEAL (Gag p17) and WPAVRERM (Nef) (data not shown). Escape variants were then driven out de novo in R2 within both FLKEKGGL (FLKEQGGL) and WPAVRERM (WPAVRKRM) (data not shown), thus attesting to the selective force of CTL in R2 during his acute infection, at least for these epitopes.
In summary, in transmitter pair 2 where the recipient is homozygous for two HLA class I alleles present in the donor (HLA-A0101 and HLA-B0801), transmission of preformed escape variants at epitopes restricted by these alleles was followed by poor HIV control as viral loads in this patient remained very high for 38 weeks until he finally agreed to take HAART (Fig. 1b).
Single-amino-acid polymorphisms can lead to escape at overlapping epitopes restricted by distinct HLA alleles. We have shown that the transmission of preformed escape variants can abolish CTL recognition in the recipient if HLA is shared. A single substitution can also lead to escape at more than one epitope with distinct HLA class I restrictions. In patient D2, we identified a K28Q substitution at the C-terminal end of the HLA-A0301-restricted Gag p17 epitope RLRPGGKKK (RLRPGGKKQ) and in the overlapping HLA-B0801-restricted GGKKKYKL epitope (GGKKQYRL) (Fig. 5). This polymorphism is at an anchor residue in both epitopes (29) and both variants showed over 80% reduction in binding affinity (Table 2). Therefore, the single K28Q polymorphism confers escape in both the HLA-A0301-restricted RLRPGGKKK and HLA-B0801-restricted GGKKKYKL epitopes.
DISCUSSION
In four HIV-1-infected homosexual couples, we identified transmission of single viruses bearing polymorphisms within epitopes restricted by the patients' HLA class I alleles a few weeks after the transmission event. We identified CTL escape variants in known donors and tracked the transmission of these viruses to the recipient, who was recruited in the early stages of acute infection. When an acutely infected patient became a transmitter himself, the precise timing of transmission was a crucial determinant of the variant viruses available to compose the infecting inoculum.
In earlier studies of sexual transmission of HIV-1 CTL escape variants (2, 15, 16, 27), little or nothing was known of the donor virus. A recent study focusing on HLA-B57/5801 alleles investigated the consequences of sexual HIV-1 transmission within identified HLA-matched and mismatched couples, although the patients were often sampled months after the transmission (27). De novo selection of variants in recipients can be difficult to exclude when sampling intervals are this long (5). Our approach, involving the identification of the transmitting couples and very close sampling (some including donor samples prior to transmission), allowed us to compare antigenic characteristics of the successful inoculum at multiple loci in donor and recipient. It is the precise pattern of antigenic variation that prevails across the transmission boundary that successful vaccines must be able to combat.
We studied internal structural genes of the virus, as these are key antigenic targets for CTL. Our analysis shows that, within the internal HIV-1 proteins, escape properties were retained during sexual transmission. This result differs from the discovery that HIV-1 resistant to autologous neutralizing antibody is infrequently represented in the viral inoculum (12). In contrast to our "immune-resistant" viruses, multidrug resistance viruses also have a low probability of transmission (7, 11, 25).
In two of our transmitter couples, the infecting inoculum had several escape variants. The process that selected these changes could have taken place years before. These escape mutations might also reflect selection in a previous transmitter, since reversion of some escape changes may be very slow (26). Inevitably, virions that carry several escape mutations will be less well recognized in the recipient where HLA class I is shared or where polymorphisms abolish recognition at overlapping restriction sites, provided they incur no increase in the fitness cost to the virus. If the recipient is homozygous for HLA class I molecules present in the donor, then the transmission of preformed escape at loci restricted by these molecules could impede viral control (10). This was the case with our D2- R2 pair.
Multiple variants, conferring escape to responses of distinct epitope restrictions, can also be transmitted by a single virion (Fig. 4 and 5). Thus, even if infection is established by a small founder population, the potential for multiple escape variants to be transmitted remains high. If HLA class I molecules are shared between partners, the recipient's ability to mount a response against infection will be impeded, particularly if escape has occurred through change at an HLA anchor residue that abolishes binding of the peptide to the HLA molecule. These changes will always confer escape properties to the infecting virus in recipients who share HLA class I. However, there are limitations to interpreting the binding data alone, due to the nature of the assays in use. Our binding results were supported by testing for CTL responses. The successful transmission of virions that carry escape mutations at several sites is likely to give these viruses a survival advantage in a new host when HLA class I is shared. The rapid progression characteristic of infantile HIV-1 infection has been attributed at least in part to the vertical transmission of preformed escape variants from the mother (and potentially the father) (17).
In contrast, escape changes that interfere with T-cell receptor recognition of the presented antigen may be specific to the individual's CTL repertoire (34, 41). In this setting the escape "phenotype" may not be transferred with the polymorphisms that conferred immune evasion in the donor, as the new host may have T-cell clones capable of accommodating the viral variants (5). There are examples of escape through failure of T-cell receptor recognition when the mutant defies all available clonal responses in numerous hosts (22, 35). When polymorphisms of this type, which do not reduce viral fitness, are transmitted, the escape phenotype would be preserved.
We studied sexual transmission of HIV-1 between patients who share HLA molecules (donor-recipient pair 2). Four transmitted escape variants were identified in epitopes restricted by shared alleles. In three of these (GGKKQYRL, YLQDQQLL, and ISERILSTF), the relevant changes occurred at an anchor residue.
Some strong responses were observed in R2 to transmitted variants, most notably DVKDTKEAL and WPAVRERM. These results indicate that transmitted anchor residue changes will lead to a loss of recognition in recipients who share the appropriate HLA class I molecule while responses to other polymorphic epitopes may be invoked. The reduction in available epitopes to which CTL can respond is likely to impede viral control but not abolish it altogether.
In a recent study describing transmission of simian immunodeficiency virus mutations between HLA-matched and mismatched rhesus monkeys, transient reversions of some variants to the wild-type sequence occurred in the recipients evoking a short-lived CTL response which reselected for the escape variants (5). We did not see reversions, and transmitted escape mutants persisted for up to 52 weeks. These findings suggest that viral fitness was not compromised by the multiple escape mutations we observed.
The transmission of some CTL escape variants can lead to their accumulation in the infected population (37, 40). Since reversion can be slow, a single escape variant may survive multiple transmission events, increasing the chance that it will eventually infect someone with the correct restricting HLA allele and enable immune escape. Maintenance of an escape variant in the infected population at high frequency may also depend on a sufficient number of encounters with the appropriate selective CTL responses. This, in turn, will be dependent on the frequency of the HLA allele in the population.
Could the acquisition of immune escape viruses diminish the ability of the infected population to control HIV and lead to faster progression rates? To answer this question, we need to know the stability of escape variants, their overall tendency to reversion if they have been selected in hosts at a fitness cost, and their "transmissability." Both transmission and reversion will be determined by the replicative fitness costs associated with CTL escape. Clearly, further work is required to survey the polymorphic patterns across large populations and so estimate such costs globally if we are to assess the consequences of the transmission of escape variants for the long-term evolution of HIV-1.
Our serial data on the acute HIV-infected donor D1 demonstrates that the pattern of variation transmitted is crucially dependent on timing, as illustrated in Fig. 3. If a patient who has just become infected then transmits, the effective inoculum may more closely reflect previous host virus selection. If the acute infection is resolving and de novo selection (and potentially reversion) has begun to play out, the transmitted virus will reflect the selection in the transmitting host. This important paradigm should be taken into account when designing anti-HIV CTL vaccines around consensus sequences, when it is attested that these vaccines will be able to induce immunity capable of "sterilizing" such variable viral challenges.
ACKNOWLEDGMENTS
This work was supported by the Wellcome Trust, the National Institutes of Health, and a Nuffield Dominion Scholarship (T.P.). R.E.P. is a founding investigator of The James Martin 21st Century School.
A.M. and C.T.T.E. contributed equally to this study.
R.E.P. and H.-T.Z. share senior authorship.
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Jefferiss Research Laboratories, Wright-Fleming Institute, Imperial College London, St. Mary's Hospital, Norfolk Place, London W2 1PG, United Kingdom
Department of Immunohaematology and Blood Transfusion, Leiden University Medical Centre, P.O. Box 9600, 2300 Leiden, The Netherlands
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, United Kingdom
ABSTRACT
Antigenic variation inherent in human immunodeficiency virus type 1 (HIV-1) virions that successfully instigate new infections transferred by sex has not been well defined. Yet this is the viral "challenge" which any vaccine-induced immunity must deal with. Closely timed comparisons of the virus circulating in the "donor" and that which initiates new infection are difficult to carry out rigorously, as suitable samples are very hard to get in the face of ethical hurdles. Here we investigate HIV-1 variation in four homosexual couples where we sampled blood from both parties within several weeks of the estimated transmission event. We analyzed variation within highly immunogenic HIV-1 internal proteins encoding epitopes recognized by cytotoxic Tlymphocytes (CTLs). These responses are believed to be crucial as a means of containing viral replication. In the donors we detected virions capable of evading host CTL recognition at several linked epitopes of distinct HLA class I restriction. When a donor transmitted escape variants to a recipient with whom he had HLA class I molecules in common, the recipient's CTL response to those epitopes was prevented, thus impeding adequate viral control. In addition, we show that even when HLA class I alleles are disparate in the transmitting couple, a single polymorphism can abolish CTL recognition of an overlapping epitope of distinct restriction and so confer immune escape properties to the recipient's seroconversion virus. In donors who are themselves controlling an early, acute infection, the precise timing of onward transmission is a crucial determinant of the viral variants available to compose the inoculum.
INTRODUCTION
A thoroughdescription of the human immunodeficiency virus type I (HIV-1) inoculum that establishes new infection is central to understanding protective immunity to this virus. Close sequence similarity in envelope has been observed in HIV-1 successfully transmitted between heterosexual African partners (12). In contrast, viral strains resistant to neutralization by antibody are infrequently transmitted (12). However, HLA class I-restricted antigenic properties of the successful infecting virus are largely unknown. Useful comparisons between "donor" HIV-1 variants and the virions that establish infection in a new host require sampling very close to the transmission event.
Here we investigate the extent to which the founding HIV-1 population represents the antigenic properties of the virus in the donor. Much evidence now supports the concept that HLA class I-restricted CD8+ T-cell responses are crucial for the control of HIV and simian immunodeficiency virus infection (8, 20, 23, 30, 38). This evidence, which implies that cytotoxic T lymphocytes (CTLs) can inhibit retroviral replication, has been used to justify vaccines that rely solely on the induction of this form of immunity for protection of na?ve subjects (6). However, the CTL response to HIV-1 selects for escape variants (9, 18, 32, 33), which can impair completely the usefulness of such vaccines (3, 4).
The transmission of CTL escape variants from mother to child has been well documented (16, 17) because transmitter pairs are readily identified. Evidence has been adduced that CTL escape variants can be transmitted sexually (2, 15, 16, 27). To make this inference, an association in chronically infected patients between the possession of a particular HLA class I allele and the presence of an escape variant was first established. When the same variant was found in recently infected patients that did not have the HLA class I molecule required for a response against the epitope, it was concluded, on reasonable grounds, that the polymorphism had been transmitted.
In the present study we set out to compare, as closely as possible, the extent to which individual virions could transfer genetically linked immune escape properties. We reasoned that immune escape was positively selected during HIV infection. Given the enormous extent of recombination in the virus, further reassortment and selection could lead to the outgrowth of single virions bearing several epitopes with immune escape properties. Once we had identified such viruses, we asked whether this "multi-immune-escape HIV" could be transmitted. If the new host shared HLA class I with the donor, then it is very possible that the recipient’s control of the virus could be undermined.
We examined the antigenic properties of HIV-1 either side of transmission in four homosexual transmitter pairs. By focusing on the immunogenic internal viral proteins, we demonstrate that CTL escape variants are readily transmitted, so that infection can be established by a virus able to simultaneously evade multiple CTL responses.
MATERIALS AND METHODS
Patient pairs. Patients attending the HIV clinic at St. Mary's Hospital, London, United Kingdom, with acute HIV infection were recruited to a study (SPARTAC) of a short-course highly active retroviral therapy (HAART). As a substudy, consenting patients were asked to identify recent sexual partners. Transmitting couples were only studied if both the recipient and the donor gave written consent.
This study was granted local research ethical approval to recruit both newly infected patients and their HIV-transmitting partners. Acute HIV infection was identified according to the following definition: previously documented HIV antibody negative within 6 months, negative antibody test with positive HIV RNA PCR, or incident infection using the Abbott detuned assay. Detailed sexual histories were taken from both partners separately.
Viral sequencing and analysis. Plasma samples containing the virus were obtained from both donor and recipient within 2 to 6 weeks of the time when acute infection was diagnosed in the recipient. HIV-1 virion RNA was reverse transcribed, amplified, cloned, and sequenced from several time points close to the time of transmission, according to a previously described protocol (31). The clonal sequencing effort was directed toward, but not limited to, the highly immunogenic gag and nef regions (1). As a precaution against contamination, all reagents were aliquoted before use, PCR amplification of the donor and recipient of each pair was spatially and temporally separated in the laboratory, and negative controls were conducted in parallel at each stage of PCR.
Phylogenetic comparison of the four transmitter pairs. Nucleotide pol sequences (1,269 bp), obtained by population sequencing and incorporating the entire protease gene and a part of the reverse transcriptase (RT) gene, were manually aligned together using the program McClade version 4.07 (28). Sequences from the four transmitter pairs; HIV-1 clade A, B, C, and D consensus sequences; and 78 pol RT sequences from acutely HIV-infected individuals attending St. Mary's Hospital, London, United Kingdom, during the period 2001 to 2004 were incorporated into the alignment. The matrix was subsequently imported into the tree building software PAUP (39), and an initial neighbor-joining (NJ) tree (36) was reconstructed under the Hasegawa-Kishino-Yano (HKY85) model of evolution (19). The NJ topology was used as a starting tree for a heuristic search for a maximum-likelihood tree (13) under the general time reversible model of nucleotide substitution (42), with proportion of invariable sites and rate heterogeneity. The parametric values of the selected model are available from the authors on request. The robustness of the NJ phylogeny was assessed by bootstrap analysis (14), with 1,000 rounds of replication.
Gamma interferon ELISPOT assay. Antigen-specific responses from patients' peripheral blood mononuclear cells (PBMCs) were measured using synthetic peptides (Invitrogen, Mimotopes, Biosynthesis) and a standard enzyme-linked immunospot (ELISPOT) assay, as previously described (24). All assays were done in duplicate. A duplicate negative control was included in all assays; the number of spots in the negative control was subtracted, and the results were normalized to give the number of spot-forming cells per million PBMCs.
Peptide binding assay. The competitive fluorescence binding assay was carried out as described previously (21). In brief, B-cell lines bearing the relevant HLA class I molecules were subjected to acid elution for 90 s at pH 3.1 (for HLA-A03-restricted peptides, a pH of 2.9 was used). The B cells were then incubated for 24 h at 2 to 8°C with an HLA-specific reference peptide conjugated to a fluorescein label and a test peptide titrated between 184 μM and 0.02 μM. After the incubation, each sample was stained with 7-aminoactinomycin D (Viaprobe; BD Biosciences) in order to exclude dead cells from subsequent analysis, fixed, and analyzed by flow cytometry. All assays were done three times. The inhibition of fluorescein-labeled reference peptide binding, through competition with the test peptide, is a measure of the binding affinity of the test peptides. Inhibition was determined as previously described (21) and expressed as the concentration that results in a 50% reduction in fluorescence (IC50). The percent reduction in binding affinity (%RBA) of a variant peptide (V), relative to the consensus (index, I) was calculated using the following formula: %RBA = [1 – (IC50I/IC50V)] x 100.
Defining immune escape variants. A polymorphism within an HLA class I-restricted epitope was defined as conferring escape if it satisfied one or more of the following criteria: (i) the epitope variant could not bind to the relevant class I allele (and hence cannot be presented to CTL), (ii) the variant was not recognized by the host (autologous) CTL although the consensus epitope was recognized, or (iii) the variant was not recognized by three sets of heterologous PBMCs. Binding assays were performed on around 90% of the analyzed variants. Wherever possible, the variants that bound to HLA were also tested for immunogenicity on autologous PBMCs by ELISPOT. The heterologous PBMCs, which can respond to the consensus epitope, were taken from HIV-infected patients and were used only when the autologous PBMCs were not available.
In the assay used for the evaluation of binding, a reduction of >99% represents a case when, at the highest concentration of the test peptide (200 μM), there was no displacement of the fluorescently labeled index peptide. In our definition of escape through lack of binding, only peptides for which binding was reduced >99% were taken as escape variants. For peptides that showed reduced (but not completely abolished) binding, immunogenic responses were also tested in order to establish the escape status of the peptide. Failure of recognition by autologous or heterologous PBMCs was recorded when there was no gamma interferon production over background (as tested by ELISPOT) at peptide concentrations of 1 μM or lower.
RESULTS
Four male homosexual patient couples were identified after diagnosis of acute HIV infection in the recipient. A pair is referred to as donor (Dn) and recipient (Rn), where n is the pair number. The times when patients were recruited and sampled are shown in Fig. 1a to d by black arrows. Orange arrows mark the time when samples were analyzed to establish the relatedness of viral sequences closest to the time of transmission. The periods of HAART are denoted by dark green bars (Fig. 1).
The close relatedness of the viruses harbored within transmission pairs was determined unambiguously by the topology of the maximum-likelihood tree (Fig. 2). For each couple, the pol RT sequences from the donor and recipient clustered tightly together. On testing the tree topology for robustness, the transmitter clusters occurred repeatedly (100% bootstrap support). The genetic distances were consistently shorter between sequences involved in transmission pairs than between unlinked sequences (average within-pair genetic distance of 0.0024 substitution/site), and transmission pairs 1 and 3 had identical sequences. All pairs, except pair 1, were infected with an HIV-1 clade B virus. Other putative transmission networks were also observed within the phylogeny, although these were not analyzed in the present study.
Clonal sequencing of the viruses present in the donors and the recipients was carried out. Consensus sequences corresponding to relevant clades were taken from the National Institutes of Health/National Institute of Allergy and Infectious Diseases databases (http://www.hiv.lanl.gov). In total, 61 epitopes were sequenced among the four transmitter pairs. Of these, 47 epitope sites showed the presence of variants. Variant and consensus peptides were synthesized and immunologically characterized by binding assays or CTL recognition or both. Variants were then scored for "escape" according to the strict definition described in the Materials and Methods section. Table 1 shows the number of epitopes in each transmitter couple that had variants that were defined as escape by virtue of not binding or lack of ELISPOT response by autologous or heterologous PBMCs. The binding characteristics of 38 of these variants and consensus peptides are reported in Table 2.
Detection of selection of escape in HIV transmitters: sample timing is crucial. D1 was recruited with primary HIV infection. He was given a short course of HAART and then experienced viral rebound (Fig. 1a). Recipient R1, who was infected by D1 (Fig. 2) almost certainly during that rebound, was sampled at the nominal time week 0 (Fig. 1a). This allowed very close HIV sequence comparisons in four samples taken from D1 before and after week 0 (Fig. 1a, D1, green inverted triangles). We found that the sequence within the p17 Gag HLA A0301-restricted epitope RLRPGGKKK in R1 (RLRPGGRKK; substitutions are underlined throughout) was identical to that detected in D1 19 weeks before R1 was first sampled, i.e., week 0 (Fig. 3). Variant RLRPGGRKT, which was not recognized by D1 and had 95% reduction in binding affinity, subsequently rose to fixation in D1. This variant was shown to be under positive selection in the donor (using the program Codeml from the PAML software package) (43). We did not find this escape variant in R1, but this is not surprising since this sequence rose from 19% to fixation in D1 20 weeks after R1 was recruited (Fig. 3, week 20). This example illustrates that the precise moment during the natural course of HIV infection at which transmission takes place can be a critical determinant as to which viral variant infects the new host (Fig. 4 and 5). This is particularly true during acute infection when the successful inoculum is rapidly adapting to a new host. Escape variants were transmitted from D1 to R1 at four other epitopes (Table 1).
Sexual transmission of HIV-1 bearing multisite CTL escape variants. We identified CTL escape variants in three of the donors (D1, D2, and D3). Some of these escape variants were transmitted (Table 1). In pair 1, 4 of 5 escape variants were transmitted; in pair 2, 8 of 10. Out of a total of 21 epitopes that were immunologically characterized, 16 encoded escape variants in the donor virus population very close (i.e., 1 to 11 weeks) to the estimated time of transmission. CTL escape variants at 12 of these sites were transmitted and detected in samples taken from the recipients when they were first identified with acute HIV infection (week 0).
Of particular interest is that viral segments bearing several linked CTL escape epitopes were identified in two donors (D1 and D2). In D1, who was infected with HIV subtype A, escape variants were present at high frequency in three epitopes of distinct HLA class I restriction within Gag p17 at 4 and 19 weeks before R1 was first recruited (Fig. 4). In D1, a single change of K to R (K26R) at the C-terminal end of the HLA-A0301-restricted KIRLRPGGK epitope led to a loss of recognition (Fig. 4, A3 inset) and a 74% reduction in HLA binding affinity (Table 2, KIRLRPGGR). For the HLA-B0801-restricted GGKKKYRL (GL8) epitope, the K26R change and R30K change (GGRKKYKL) resulted in failure of recognition by HLA-B0801-restricted T cells specific for the GL8 epitope (Fig. 4, B8 inset). An additional instance of escape in D1 was observed in the HLA-A0101-restricted GTEELRSLY epitope: the GTEELRSLF variant found in D1 was not recognized by heterologous PBMCs at low peptide concentrations (Fig. 4, A1 inset). Binding assays showed that the change of Y to F (Y79F) had reduced the HLA-A0101 binding affinity of the peptide by 98% (Table 2).
Therefore, in D1 we detected single virions bearing polymorphisms at three epitopes of distinct HLA class I restriction, all of which individually conferred immune escape. Virions containing the identical three linked escape variants were also found in all of the sequenced clones in R1 during acute HIV infection (week 0) and were propagated for 52 weeks (Fig. 4). According to a detailed clinical history of each patient, transmission from D1 to R1 is most likely to have occurred 3 to 6 weeks prior to week 0. There was no evidence of reversion of these polymorphisms in R1 during this period.
Transmission of escape variants abolished CTL recognition in the recipient who shared HLA alleles with his donor. For donor-recipient pair 2, who share HLA-A0101 and HLA-B0801, transmission is likely to have occurred 4 to 9 weeks prior to week 0 (the time R2 was first sampled) (Fig. 5). Variants were detected at four epitopes in D2 on a continuous sequence encoding Gag p17, collected from samples 11 and 22 weeks before R2 was recruited (RLRPGGKKQ, GGK KQYRL, GSEEIKSLY, and DVKGTKEAL). These variants were not recognized by CTL in ELISPOT assays (Fig. 5, insets).
A change of K to Q (K28Q) at the C-terminal end of the HLA-A0301-restricted RLRPGGKKK epitope was not recognized, and the variant RLRPGGKKQ had a reduced binding affinity of >99% (Table 2). As well as directly leading to escape in the RLRPGGKKK epitope, this change has been shown to confer escape in the neighboring HLA-A3-restricted KIRLRPGGK epitope by interfering with its processing (2).
The K28Q and the K30R change of the HLA-B0801-restricted overlapping GGKKKYKL epitope are associated with a loss of recognition. The K28Q polymorphism lies at an anchor residue in HLA-B0801-restricted epitopes, and the K30R change can lead to antagonism of the GGKKKYKL response (35). The GGKKQYRL variant peptide sequence, detected in both D2 and R2, had an 82% reduction in binding affinity assays (Table 2).
Variants at the HLA-A0101-restricted GSEELRSLY (variant GSEEIKSLY) and HLA-B0801-restricted EVK DTKEAL (variant DVKGTKEAL) epitopes were also tested in ELISPOT assays. The GSEEIKSLY variant showed a 44% reduction in binding affinity (Table 2). Neither variant was recognized in ELISPOT assays (Fig. 5, A1 and B8 insets). The escape variant DVKGTKEAL was not transmitted. As in D1, individual virions encoding immune escape variants at several linked sites were found in Gag p17 in D2. With the exception of the EVKDTKEAL epitope, all Gag p17 substitutions responsible for escape were present at high frequency in R2.
Escape variants were transmitted from D2 to R2 at an additional two sites, HLA-B0801-restricted YLQDQQLL in Env gp41 and HLA-A0101-restricted ISERILSTF in Rev. Both of these variants had over 90% reduction in binding affinity (Table 2).
Despite the absence of a detectable response to escape variants encoded at four epitopes, R2 mounted a response to the HLA-B0801-restricted FLKEKGGL Nef epitope, and the HLA-B0801-restricted variants DVKDTKEAL (Gag p17) and WPAVRERM (Nef) (data not shown). Escape variants were then driven out de novo in R2 within both FLKEKGGL (FLKEQGGL) and WPAVRERM (WPAVRKRM) (data not shown), thus attesting to the selective force of CTL in R2 during his acute infection, at least for these epitopes.
In summary, in transmitter pair 2 where the recipient is homozygous for two HLA class I alleles present in the donor (HLA-A0101 and HLA-B0801), transmission of preformed escape variants at epitopes restricted by these alleles was followed by poor HIV control as viral loads in this patient remained very high for 38 weeks until he finally agreed to take HAART (Fig. 1b).
Single-amino-acid polymorphisms can lead to escape at overlapping epitopes restricted by distinct HLA alleles. We have shown that the transmission of preformed escape variants can abolish CTL recognition in the recipient if HLA is shared. A single substitution can also lead to escape at more than one epitope with distinct HLA class I restrictions. In patient D2, we identified a K28Q substitution at the C-terminal end of the HLA-A0301-restricted Gag p17 epitope RLRPGGKKK (RLRPGGKKQ) and in the overlapping HLA-B0801-restricted GGKKKYKL epitope (GGKKQYRL) (Fig. 5). This polymorphism is at an anchor residue in both epitopes (29) and both variants showed over 80% reduction in binding affinity (Table 2). Therefore, the single K28Q polymorphism confers escape in both the HLA-A0301-restricted RLRPGGKKK and HLA-B0801-restricted GGKKKYKL epitopes.
DISCUSSION
In four HIV-1-infected homosexual couples, we identified transmission of single viruses bearing polymorphisms within epitopes restricted by the patients' HLA class I alleles a few weeks after the transmission event. We identified CTL escape variants in known donors and tracked the transmission of these viruses to the recipient, who was recruited in the early stages of acute infection. When an acutely infected patient became a transmitter himself, the precise timing of transmission was a crucial determinant of the variant viruses available to compose the infecting inoculum.
In earlier studies of sexual transmission of HIV-1 CTL escape variants (2, 15, 16, 27), little or nothing was known of the donor virus. A recent study focusing on HLA-B57/5801 alleles investigated the consequences of sexual HIV-1 transmission within identified HLA-matched and mismatched couples, although the patients were often sampled months after the transmission (27). De novo selection of variants in recipients can be difficult to exclude when sampling intervals are this long (5). Our approach, involving the identification of the transmitting couples and very close sampling (some including donor samples prior to transmission), allowed us to compare antigenic characteristics of the successful inoculum at multiple loci in donor and recipient. It is the precise pattern of antigenic variation that prevails across the transmission boundary that successful vaccines must be able to combat.
We studied internal structural genes of the virus, as these are key antigenic targets for CTL. Our analysis shows that, within the internal HIV-1 proteins, escape properties were retained during sexual transmission. This result differs from the discovery that HIV-1 resistant to autologous neutralizing antibody is infrequently represented in the viral inoculum (12). In contrast to our "immune-resistant" viruses, multidrug resistance viruses also have a low probability of transmission (7, 11, 25).
In two of our transmitter couples, the infecting inoculum had several escape variants. The process that selected these changes could have taken place years before. These escape mutations might also reflect selection in a previous transmitter, since reversion of some escape changes may be very slow (26). Inevitably, virions that carry several escape mutations will be less well recognized in the recipient where HLA class I is shared or where polymorphisms abolish recognition at overlapping restriction sites, provided they incur no increase in the fitness cost to the virus. If the recipient is homozygous for HLA class I molecules present in the donor, then the transmission of preformed escape at loci restricted by these molecules could impede viral control (10). This was the case with our D2- R2 pair.
Multiple variants, conferring escape to responses of distinct epitope restrictions, can also be transmitted by a single virion (Fig. 4 and 5). Thus, even if infection is established by a small founder population, the potential for multiple escape variants to be transmitted remains high. If HLA class I molecules are shared between partners, the recipient's ability to mount a response against infection will be impeded, particularly if escape has occurred through change at an HLA anchor residue that abolishes binding of the peptide to the HLA molecule. These changes will always confer escape properties to the infecting virus in recipients who share HLA class I. However, there are limitations to interpreting the binding data alone, due to the nature of the assays in use. Our binding results were supported by testing for CTL responses. The successful transmission of virions that carry escape mutations at several sites is likely to give these viruses a survival advantage in a new host when HLA class I is shared. The rapid progression characteristic of infantile HIV-1 infection has been attributed at least in part to the vertical transmission of preformed escape variants from the mother (and potentially the father) (17).
In contrast, escape changes that interfere with T-cell receptor recognition of the presented antigen may be specific to the individual's CTL repertoire (34, 41). In this setting the escape "phenotype" may not be transferred with the polymorphisms that conferred immune evasion in the donor, as the new host may have T-cell clones capable of accommodating the viral variants (5). There are examples of escape through failure of T-cell receptor recognition when the mutant defies all available clonal responses in numerous hosts (22, 35). When polymorphisms of this type, which do not reduce viral fitness, are transmitted, the escape phenotype would be preserved.
We studied sexual transmission of HIV-1 between patients who share HLA molecules (donor-recipient pair 2). Four transmitted escape variants were identified in epitopes restricted by shared alleles. In three of these (GGKKQYRL, YLQDQQLL, and ISERILSTF), the relevant changes occurred at an anchor residue.
Some strong responses were observed in R2 to transmitted variants, most notably DVKDTKEAL and WPAVRERM. These results indicate that transmitted anchor residue changes will lead to a loss of recognition in recipients who share the appropriate HLA class I molecule while responses to other polymorphic epitopes may be invoked. The reduction in available epitopes to which CTL can respond is likely to impede viral control but not abolish it altogether.
In a recent study describing transmission of simian immunodeficiency virus mutations between HLA-matched and mismatched rhesus monkeys, transient reversions of some variants to the wild-type sequence occurred in the recipients evoking a short-lived CTL response which reselected for the escape variants (5). We did not see reversions, and transmitted escape mutants persisted for up to 52 weeks. These findings suggest that viral fitness was not compromised by the multiple escape mutations we observed.
The transmission of some CTL escape variants can lead to their accumulation in the infected population (37, 40). Since reversion can be slow, a single escape variant may survive multiple transmission events, increasing the chance that it will eventually infect someone with the correct restricting HLA allele and enable immune escape. Maintenance of an escape variant in the infected population at high frequency may also depend on a sufficient number of encounters with the appropriate selective CTL responses. This, in turn, will be dependent on the frequency of the HLA allele in the population.
Could the acquisition of immune escape viruses diminish the ability of the infected population to control HIV and lead to faster progression rates? To answer this question, we need to know the stability of escape variants, their overall tendency to reversion if they have been selected in hosts at a fitness cost, and their "transmissability." Both transmission and reversion will be determined by the replicative fitness costs associated with CTL escape. Clearly, further work is required to survey the polymorphic patterns across large populations and so estimate such costs globally if we are to assess the consequences of the transmission of escape variants for the long-term evolution of HIV-1.
Our serial data on the acute HIV-infected donor D1 demonstrates that the pattern of variation transmitted is crucially dependent on timing, as illustrated in Fig. 3. If a patient who has just become infected then transmits, the effective inoculum may more closely reflect previous host virus selection. If the acute infection is resolving and de novo selection (and potentially reversion) has begun to play out, the transmitted virus will reflect the selection in the transmitting host. This important paradigm should be taken into account when designing anti-HIV CTL vaccines around consensus sequences, when it is attested that these vaccines will be able to induce immunity capable of "sterilizing" such variable viral challenges.
ACKNOWLEDGMENTS
This work was supported by the Wellcome Trust, the National Institutes of Health, and a Nuffield Dominion Scholarship (T.P.). R.E.P. is a founding investigator of The James Martin 21st Century School.
A.M. and C.T.T.E. contributed equally to this study.
R.E.P. and H.-T.Z. share senior authorship.
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