TRABAJO PRÁCTICO VIROLOGÍA Artículo N°1: “Unusual Naturally Occurring Humoral and Cellular Mutated Epitopes of Hepatitis B Virus in a Chronically Infected Argentine Patient with Anti-HBs Antibodies” de Cuestas y cols. 1) Resuma el caso clínico del paciente presentado en el artículo. 2) ¿Qué métodos de diagnóstico se emplearon en el artículo? Clasifíquelos en directos e indirectos. 3) ¿Qué mecanismo de evasión viral se describe en el artículo? 4) ¿Considera que los hallazgos y las conclusiones arrojadas por este artículo científico son importantes desde el punto de vista de la salud pública? ¿Por qué? Artículo N°2: “Disease progression due to dual infection in an HLA-B57-positive asymptomatic long-term nonprogressor infected with a nef-defective HIV-1 strain” de Braibant y cols. 1) Resuma el caso clínico del paciente presentado en el artículo. 2) ¿Qué métodos de diagnóstico se emplearon en el artículo? Clasifíquelos en directos e indirectos. 3) ¿Qué factores virales y genéticos del hospedador le conferían protección frente a la infección viral al paciente del artículo? ¿Por qué? 4) ¿Qué mecanismo de evasión viral se describe en el artículo? 5) ¿Considera que los hallazgos y las conclusiones arrojadas por este artículo científico son importantes para tener en cuenta en la generación de una vacuna anti-HIV? ¿Por qué?
Transcript
TRABAJO PRÁCTICO VIROLOGÍA
Artículo N°1: “Unusual Naturally Occurring Humoral and Cellular Mutated Epitopes of
Hepatitis B Virus in a Chronically Infected Argentine Patient with Anti-HBs Antibodies” de Cuestas y cols.
1) Resuma el caso clínico del paciente presentado en el artículo. 2) ¿Qué métodos de diagnóstico se emplearon en el artículo? Clasifíquelos en directos e indirectos. 3) ¿Qué mecanismo de evasión viral se describe en el artículo? 4) ¿Considera que los hallazgos y las conclusiones arrojadas por este artículo científico son importantes desde el punto de vista de la salud pública? ¿Por qué?
Artículo N°2: “Disease progression due to dual infection in an HLA-B57-positive
asymptomatic long-term nonprogressor infected with a nef-defective HIV-1 strain” de Braibant y cols.
1) Resuma el caso clínico del paciente presentado en el artículo.
2) ¿Qué métodos de diagnóstico se emplearon en el artículo? Clasifíquelos en directos e indirectos. 3) ¿Qué factores virales y genéticos del hospedador le conferían protección frente a la infección viral al paciente del artículo? ¿Por qué? 4) ¿Qué mecanismo de evasión viral se describe en el artículo? 5) ¿Considera que los hallazgos y las conclusiones arrojadas por este artículo científico son importantes para tener en cuenta en la generación de una vacuna anti-HIV? ¿Por qué?
Unusual Naturally Occurring Humoral and Cellular Mutated Epitopesof Hepatitis B Virus in a Chronically Infected Argentine
Patient with Anti-HBs AntibodiesMarıa L. Cuestas,1 Veronica L. Mathet,1 Vanesa Ruiz,1 Marıa L. Minassian,1 Cintia Rivero,1
Andrea Sala,2 Daniel Corach,2 Analıa Alessio,3 Marcia Pozzati,3 Bernardo Frider,3and Jose R. Oubina1*
Dto. Microbiologıa, Fac. de Medicina, UBA,1 Servicio de Huellas Digitales Geneticas y Catedra de Genetica y Biologıa Molecular,Fac. de Farmacia y Bioquımica, UBA,2 and Div. Clınica Medica-Hepatologıa, Hospital Argerich,3 Buenos Aires, Argentina
Received 10 January 2006/Returned for modification 11 February 2006/Accepted 10 March 2006
Serum hepatitis B virus (HBV) DNA was extracted from a chronically infected patient with cocirculation ofhepatitis B surface antigen (HBsAg) and anti-HBs antibodies. Direct PCR and clone-derived sequences of theS and overlapped P genes were obtained. DNA sequences and phylogenetic analysis ascribed this isolate togenotype A (serotype adw2). Five of six HBV DNA clones exhibited point mutations inside and outside themajor hydrophilic region, while the sixth clone exhibited a genotype A “wild-type” amino acid sequence.Observed replacements included both humoral and/or cellular (major histocompatibility complex class I[MHC-I] and MHC-II) HBV mutated epitopes, such as S45A, P46H, L49H, C107R, T125A, M133K, I152F,P153T, T161S, G185E, A194T, G202R, and I213L. None of these mutants were individually present within agiven clone. The I213L replacement was the only one observed in the five clones carrying nonsynonymousmutations in the S gene. Some of the amino acid substitutions are reportedly known to be responsible for theemergence of immune escape mutants. C107R replacement prevents disulfide bonding, thus disrupting the firstloop of the HBsAg. Circulation of some of these mutants may represent a potential risk for the community,since neither current hepatitis B vaccines nor hyperimmune hepatitis B immune globulin are effectively preventthe liver disease thereto associated. Moreover, some of the recorded HBsAg variants may influence theaccuracy of the results obtained with currently used diagnostic tests.
In addition to acute infections, hepatitis B virus (HBV) maybe associated with chronic hepatitis, liver cirrhosis, and hepa-tocellular carcinoma. Both host- and virus-related factors havebeen involved in viral persistence. Among the latter, viral vari-ability is regarded as a key strategy in the attempt to avoid bothhumoral and cellular immune responses (reviewed in reference33). Several reports have documented the appearance of es-cape mutants from protective anti-HBs antibodies as well asfrom cytotoxic T lymphocyte-specific clones. Spontaneous mu-tations at T-cell receptor contact sites within individual viralepitopes can abrogate or antagonize the recognition of thecorresponding wild-type epitope. Such mutations may contrib-ute to viral persistence (antagonism for T cell receptor),though its clinical relevance appears to be limited (31, 41;reviewed in references 10, 32, and 33).
The HBV envelope is composed of host-derived lipids andthree related proteins: the large (LHBsAg), middle (MHBsAg),and small (SHBsAg) proteins (24).The S protein is the majorone; it is coded for by the S gene and made up of 226 aminoacids (aa) (41). As observed in Fig. 1, all three envelopeproteins contain the SHBsAg antigenic sites. Antibodiesdirected against HBsAg confer protective immunity and arecrucial for HBV clearance in patients as well as being mark-
ers associated with both active and passive prophylactic pro-tection (31).
The exact three-dimensional structure of the S protein isunknown (3). However, it is widely accepted that HBs antige-nicity is mainly dependent on the conformation of the a deter-minant, placed within the major hydrophilic region (MHR) ofthe HBsAg (Fig. 1). The MHR encompasses amino acids 101to 160 of the HBsAg and is exposed on the surface of bothvirions and subviral particles (Fig. 1, top). This region is highlyimmunogenic and is potentially under selective pressure of theimmune system. The MHR includes a complex conformationalregion named the a determinant (Fig. 1) which is dependent ondisulfide bonding among highly conserved Cys residues. It isthought that the a determinant consists of two loops main-tained by disulfide bridges between Cys 107 and 138 and Cys139 and 147 (Fig. 1, top). A large proportion of serum anti-HBs is directed against this major determinant, the main neu-tralization epitope. Amino acid substitutions within the a de-terminant can lead to conformational changes, which in turn,can affect the binding of the neutralizing antibodies (6, 27).However, the clinical significance of most of these mutants isstill uncertain (37). The epidemiological importance of suchHBs mutants is supported by reports from Taiwan, wherethe HBV vaccination program was associated with an in-creased prevalence of HBsAg mutants, concurrent with a 10-fold decrease in the HBs carrier rate in children (18). Thesedata would imply that the selective pressure induced by vacci-nation might promote the emergence of vaccine-resistantstrains (40). However, a recent study carried out in Pacific
Island countries has suggested that vaccine escape variants arenot an important cause for failing to prevent HBV transmis-sion in this geographical area (1).
Some S mutants can affect the HBV polymerase proteinsequence due to the overlapping nature of both open readingframes (ORFs) (Fig. 1). As a result, mutations in the S genemay or may not affect the catalytic domain of the polymerase
gene and vice versa. Mutations within the a determinant andthe corresponding fragment of the viral polymerase (A and Bregions within the reverse transcriptase [RT] domain) (Fig. 1)are more frequently observed among chronic carriers with anti-HBc antibodies as the only serological marker for HBV com-pared with HBsAg-positive patients (39).
Mutations outside the MHR (Fig. 1), around codons 44 to
FIG. 1. Schematic representation of the S protein (positions 101 to 160), including the first and second loops, according to the second modelproposed by Carman et al. (6). Shadowed circles represent Cys residues. Disulfide bonds are shown by double bars. Mutated residues as detectedin clone sequences are indicated by arrows pointing to the respective amino acid replacement. A representation of the overlapped S and P genesand the genomic region amplified by PCR is shown. S gene-encoded envelope proteins (large, middle, and small HBsAg) are depicted at thebottom. The MHR is shown between aa 101 and 160 and is also indicated as a thicker zone on lines corresponding to S, pre-S2, and pre-S1 proteins.The a determinant is placed inside the MHR. The catalytic site of the HBV RT is indicated with an arrow pointing to the YMDD amino acidicregion of the RT C domain.
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49 and 152 to 213 of the S protein, were also described, thusaffecting several B-cell and major histocompatibility com-plex class I (MHC-I) and MHC-II T-cell epitopes that mightbe associated with viral persistence (2, 8, 14, 24, 30, 36).
In this study, we report further evidence of chronic hepatitisB infection despite the cocirculation of usually protective anti-HBs antibodies. Moreover, the simultaneous detection of mu-tated S- and P-derived MHC-I and MHC-II epitopes in agenotype A HBV-infected patient is described.
MATERIALS AND METHODS
Patient. A 43-year-old Argentine male (C) with chronic hepatitis B was stud-ied. He had not been vaccinated against HBV and had no known risk factors forcontracting viral hepatitis, such as intravenous drug abuse, transfusions, trans-plants, sexual preferences, surgeries, and/or prolonged stay in areas of HBVendemicity. However, his sexual partner (unvaccinated for HBV) proved positivefor anti-HBs, anti-HBe, and total anti-HBc antibodies and negative for HBsAg.She received a blood transfusion in 1986, the putative source of a persistenthepatitis C virus (HCV) infection. Symptoms related to viral hepatitis wereabsent to date. None of her three descendants (at present 20, 16, and 14 yearsold, respectively) exhibit serologic markers of HBV infection. The infectionsource of any member of the couple remains unknown.
In June 2002, the patient received medical care at Argerich Hospital, in thecity of Buenos Aires, exhibiting reactive arthritis, myalgia, elevated serumtransaminases (aspartate aminotransferase, 98 IU/ml; alanine aminotransferase,242 IU/ml) and seropositivity for HBsAg, HBeAg, total anti-HBc, and anti-HBsantibodies (33.6 mIU/ml) and negative for anti-HBe antibodies. Serologicalmarkers for HCV and human immunodeficiency virus were negative.
The simultaneous seropositivity for HBsAg and anti-HBs antibodies was eval-uated three times and independently performed in two laboratories.
Clinical symptoms disappeared after 3 weeks, and the patient remainedasymptomatic thereafter. A liver biopsy was performed in June 2003, and his-tology showed chronic hepatitis with moderate necroinflammatory activity, fi-brosis, and steatosis. In February 2004, the viral load was 1.87 � 105 genomes permilliliter of serum (Amplicor HBV monitor; Roche Diagnostic Systems, Branch-burg, NJ). Serum transaminases remained still high (aspartate aminotransferase,181 IU/ml; alanine aminotransferase, 406 IU/ml). The patient had not receivedtreatment during the follow-up up to October 2004. Taking into account thesimultaneous detection of both HBsAg and anti-HBs antibodies, the raisedtransaminase levels, and the circulating HBV DNA, the patient was then re-ferred to the Hepatitis Laboratory, Department of Microbiology, Faculty ofMedicine, University of Buenos Aires, to perform molecular biology studies.After that, the patient started treatment with pegylated alpha 2b interferon (120�g of polyethylene glycol-INTRON weekly for 6 months). Initial falls in bothviral load and serum transaminases were observed. After 1 year, the patient isstill under clinical and virological evaluation. Serum transaminase levels remainstill elevated.
Written informed consent was provided by the patient to carry out all thestudies herein described. Results shown in this study correspond to a bloodsample obtained before interferon treatment.
Serology. Serological tests for HBsAg, anti-HBs, HBeAg, anti-HBe, and totalanti-HBc antibodies were carried out by using commercially available standardmicroparticle enzyme immune assay procedures (AxSYM; Abbott Laboratories,Ill.). Serological tests for human immunodeficiency virus and HCV were alsoperformed by following the manufacturer’s instructions (Abbott).
PCR amplification. Viral DNA was extracted from 100 �l serum by using aDNA extraction kit (Macherey-Nagel, Germany) according to the manufactur-er’s instructions. PCR amplification of the S gene (541 bp spanning nucleotidepositions 256 to 796) was performed by following a previously described protocol(22, 25) using Taq DNA polymerase and primers P7 (sense, 5�-GTG GTG GACTTC TCT CAA TTT TC-3�) and P8 (antisense, 5�-CGG TA [A/T] AAA GGGACT CA [A/C] GAT-3�). PCR products encompass an overlapping region ofboth S and P genes (Fig. 1). Kwok and Higuchi rules (21) were strictly followed.Their effectiveness was routinely assessed by testing known negative serum sam-ples and reagent controls.
Cloning. The PCR products corresponding to the HBV S gene from thepatient serum were cloned into the pGEM-T Easy vector system (Promega,Madison, WI) according to the manufacturer’s instructions. Transformed Esch-erichia coli colonies were initially screened for the HBV S gene DNA by PCR,
and the plasmid DNA from PCR-positive colonies was purified by using themodified alkaline lysis-polyethylene glycol precipitation procedure.
DNA sequencing. DNA from each clone and from the PCR product obtainedfrom serum was bidirectionally sequenced by using Big-Dye termination chem-istry (Applied Biosystems) with P7 and P8 primers, and the sequencing productswere analyzed with an ABI 3100-Avant (Applied Biosystems) capillary-basedautomatic sequencer. The PCR-derived sequence was obtained from three in-dependent sequencing reactions.
The potential Taq-dependent DNA misincorporation rate was investigated bybidirectionally sequencing PCR products derived from a GB virus C/hepatitis Gvirus clone as previously described (25). Briefly, a cDNA fragment of 325 bp wasamplified in triplicate and sequenced with specific forward and reverse primers.
HBV phylogenetic analysis. Initially, an alignment of the DNA sequencesobtained from clones as well as from the PCR product and from selected isolatesdeposited in the GenBank database ascribed to each of the eight HBV genotypeswas carried out. The phylogenetic analysis was subsequently performed by usingseveral programs included within the Phylip package (version 3.5.c).
Nucleotide sequence accession number. Nucleotide sequences have been de-posited in GenBank under the following accession numbers: clones 1 to 6,DQ350602 to DQ350607, respectively; PCR-derived sequence, DQ350608.
RESULTS
HBV cloning, sequencing, and phylogenetic analysis. Phylo-genetic analysis of the DNA sequences corresponding to theHBV S gene obtained from six clones, as well as from PCRproducts, assigned genotype A to this isolate (data not shown).All 16 amino acids that determine the adw2 subtype of theHBsAg (4) proved conserved when the DNA sequence wastranslated.
Hepatitis B surface variants were detected in 5 of the 6analyzed clones, whereas the remaining one exhibited a wild-type virus. Neither insertions nor deletions were recordedwithin the S gene. Nonsynonymous nucleotide point mutationswere associated with replacements at codons 45, 46, 49, 107,125, 133, 152, 153, 161, 185, 194, 202, and 213, some of themalso affecting the overlapping polymerase ORF (Fig. 1; Table1). A summary of the involvement of such substitutions in B-,MHC-I-, and MHC-II-restricted epitopes is depicted in Fig. 3.
Analysis of the HBV S ORF in the 5 clones with surfacevariants. The amino acid sequences of the S antigen as de-duced from the DNA sequence analysis of these variantsproved diverse. As shown in Table 1, all mutated clones exhib-ited at least one substitution within the HBsAg, either insideand/or outside the MHR, in addition to the amino acid changeI213L. Mutations within the S gene were more frequentoutside than inside this region. Most amino acid replace-ments within the a determinant occurred in the first loop(Fig. 1, top). Some of these substitutions profoundly af-fected the hydrophilicity profile of the S protein, as observedin Fig. 2.
As shown in Table 1 and Fig. 3, three of six clones exhibitedamino acid substitutions within a reportedly known region(positions 28 to 51) encompassing a class I T-cell epitope of theHBsAg (10, 23, 36), as were S45A (two clones), P46H, andL49H. Moreover, as observed in Fig. 3, two clones exhibitedamino acid exchanges in class II T helper epitopes (positions136 to 155) (2), as were I152F and P153T, while 5 clonesshowed the I213L replacement in another class II T-cellepitope (positions 213 to 226) (2).
Five clones (Table 1 and Fig. 3) showed variations withinB-cell epitopes (HBsAg 44 to 49, 115 to 155, 124 to 147, and160 to 207) (7, 14, 30).
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One clone exhibited Arg instead of Cys at position 107(C107R) (Fig. 1, top), which avoids first loop formation ac-cording to the last model of Carman et al. (6).
Analysis of the overlapping HBV polymerase ORF in the 5clones with surface variants. As the P gene is in the �1 readingframe with respect to the overlapping S gene, the deducedamino acid sequence of the corresponding fragment of theviral polymerase was also examined. This fragment representsan important part of the RT domain, including regions B andC, which may be associated with resistance to nucleoside ana-logues such as lamivudine. The YMDD motif, which is essen-tial for the enzymatic activity of many RTs, was conserved inthe 5 clones showing variations in the surface antigen. Severalresidues of the HBV DNA polymerase fragment that overlapswith the ORF encoding HBsAg were affected by surface variantsin this study. Replacement at position 569 (I569F) was detected inthe 5 clones showing variations in the S antigen but was involvedin neither B- nor T-cell epitopes. This position corresponds to theamino acid 213 of HBsAg. A single clone exhibited a stop codonat position 489 (i.e., position 133 of the S antigen).
One clone showed an amino acid exchange in the 455 to 463MHC-I-restricted epitope (L463S) (Fig. 3). The two clonesmentioned above exhibiting amino acid substitutions in theMHC-II-restricted 136 to 155 S epitope (I152F and P153T)also showed the corresponding substitutions (H508L andP509H) in both the MHC-II 503 to 517 Pol epitope (26) andthe nested MHC-I 504 to 512 Pol epitope (Fig. 3) (26). To
date, no B-cell Pol epitopes have been reported so far withinthe PCR-amplified region herein described (43).
Direct sequencing of PCR-derived products: analysis ofHBV S and polymerase ORF. As expected after sequence anal-ysis of the clones, mutations were also detected within the PCRproducts at positions 45 (S45A) and 213 (I213L) of the S gene.The observed nonsynonymous mutations corresponded tochanges at the first base of both codons: a TCA3GCA trans-version for the former replacement, and an ATA3TTA trans-version for the latter. Coincidentally, such replacements wererepresented within the P ORF (I401S and I569F) as well. Incontrast, none of the remaining substitutions observed as mu-tations within the genome of a given clone (of six studied)proved detectable within the sequence of PCR products. Noevidence of mixed HBV populations at a given nucleotideposition was recorded throughout the analyzed genomic re-gion. Twelve nucleotides could not be faithfully assigned, al-though all of them were uniformly the same base in the cor-responding position in all 6 analyzed clones. Nevertheless,none of these unassigned nucleotides in the PCR-derived se-quence were located at mutated positions as observed in thestudied clones. Thus, PCR sequence ambiguities were un-doubtedly solved by gene cloning.
DISCUSSION
In this study, a total of 13 S gene nonsynonymous amino acidexchanges (S45A, P46H, L49H, C107R, T125A, M133K,I152F, P153T, Y161S, G185E, A194T, G202R, and I213L)(Table 1) were detected in five of six analyzed clones in apatient who exhibited chronic active hepatitis despite the pres-ence of (usually neutralizing) anti-HBs antibodies with cocir-culating HBsAg. The PCR-derived sequence confirmed bothS45A and I213L substitutions. Interestingly, when the consen-sus sequence was obtained (data not shown), the S45A replace-ment could not be detected, reflecting the fact that only 2 of 6clones exhibited the mutated base, therefore emphasizing theintrinsic value of sequencing PCR products. It seems likely thatif more clones had been sequenced, the consensus sequenceand the PCR-derived sequence could have been identical.Since S45A exchange is placed at both a T- and a B-cellepitope (positions 44 to 49), this replacement might be crucialfor immune escape, as the case might be for P46H and L49Has well.
The remaining substitutions mentioned above do not seemto be PCR artifacts, as we have considered the following items:(i) the reportedly known misincorporation rate of Taq DNApolymerase per polymerized nucleotide is estimated within therange of 12.1 � 10�5 (15) to 8 � 10�6 (11); (ii) no mutationswere observed when an unrelated GB virus/hepatitis G viruscDNA clone was amplified in triplicate and bidirectionallysequenced (975 nucleotides examined twice) (25), and (iii) theshort length (541 bp) of the amplified products. Moreover, theprobability, P, that the drastic C107R substitution (see below)would have merely occurred by chance is equal to 0.0005 (1/497 � 1/4), where 497 is the number of Taq-polymerized nu-cleotides (between primers) and 4 is the number of possiblenucleotides at a given position.
This study shows a persistent infection with a mixture of botha so-called “wild-type” (1 clone) and S gene variants (5 clones)
TABLE 1. Amino acid changes in the HBsAg and the overlappingpolymerase open reading frame of the 5 clones showing
a Amino acid positions of both HBsAg and polymerase reflect the correspond-ing number for each residue at the same location of both out-of-frame overlap-ping ORFs, as represented in Fig. 1A; i.e., position 107 of HBsAg correspondsto position 463 of polymerase and likewise for the rest of the correspondingpositions. Note that most substitutions in HBsAg affect the polymerase aminoacid sequence and vice versa.
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(Table 1). As the reported patient had never received hepatitis Bimmune globulin immunoprophylaxis or the HBsAg vaccine orbeen on antiviral therapy, it is suggested that surface variantsmight have emerged or been selected despite the host immunepressure. The source of his infection remains unknown at present.However, a putative role of his partner cannot be ruled out due toan eventual past occult infection (i.e., HBV DNA positive by PCRand HBsAg negative).
According to Ogura et al. (28), mutations within the a de-terminant during the natural course of infection are predom-inantly observed within the first loop (aa 107 to 138) (5, 6, 35),whereas those induced under immune pressure due to activeand/or passive immunization are more frequently observedwithin the second loop (aa 139 to 147) (16, 29, 31, 34).
In this study, no amino acid changes occurred within thesecond loop, whereas three amino acid substitutions were ob-served within the first loop (C107R, T125A, and M133K) (Fig.1, top). Such amino acid replacements might significantly/dras-tically affect the loop structure and its hydrophilicity profile(Fig. 2), since Cys, Thr, and Met residues exhibit an accessi-bility index of �10, 7.1, and 1.9, respectively, in contrast to thatassigned for Arg, Ala, and Lys residues (9.8, 2.7, and 10, re-spectively). Furthermore, C107 is known to participate in thedisulfide bonding of the S protein (Fig. 1) and is indispensable
for secretion of the 20-nm particles (24). Its substitution by alarge and positively charged amino acid, such as Arg, impliesthe impossibility of maintaining the correct conformation ofthe a determinant, thus preventing binding of neutralizing an-tibodies. A significant involvement of Cys replacements in theemergence of variants despite the presence of anti-HBs anti-bodies has recently been described (25), although it is the firsttime that this particular C107R substitution (chromatogramavailable upon request) is observed.
Mutations outside the MHR are frequent and tend to clusterin two regions around codons 44 to 49 and 152 to 213. The firstregion contains both an MHC-I-restricted T-cell epitope and aB-cell epitope, whereas the second region, at least up to aminoacid 207, exhibits both MHC-II T helper epitopes (positions136 to 155, 163 to 174, and 213 to 226) (2) as well as B-cellepitopes (14, 30). It was also reported (14) that changes withinthis second region, located immediately downstream of the adeterminant, may alter the conformation of this immunogenicdeterminant. In agreement with this notion, Hou et al. (17)showed that amino acid insertions and deletions in this regionabolish the binding to anti-HBs antibodies.
Considering that only a single mutation was observed in all fivemutated clones within the surface antigen (I213L), a GenBanksearch was carried out to determine its frequency among
FIG. 2. Hydrophilicity patterns obtained for HBV clones. A partial analysis of the S protein (amino acid positions 101 to 160) encompassingthe MHR is shown (BioEdit program, 1999). The amino acid sequence corresponding to the wild-type (wt) sequence (as observed in clone 6) isdrawn in red. Profiles depicting mutated clones are shown in blue, green, brown, black, and dark blue and correspond to clones 1, 2, 3, 4, and 5,respectively.
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worldwide HBV isolates. After alignment of genotype A Sgene partial sequences (n � 35, including V00866, M57663,X02763, X51970, Z35717, E00010, X70185, Z72478, L13994,and S50225) (data not shown), it was observed that a Leuresidue at position 213 was extremely unusual, since only oneof them exhibited such replacement. Moreover, the observa-tion of a Leu residue at position 213 has been reported in 1 of37 (adw2) HBV-infected unvaccinated individuals, although itwas observed in 1 ayw1 isolate (16). This intriguingly predom-inant substitution (observed in 5 of 6 clones of the HBV ge-notype A isolate herein reported) might suggest a putative rolewithin the T helper epitope to evade CD4� (HBV epitope)-specific cells. However, it is reportedly known that amino acidslocated at the extreme of a given MHC-II epitope have little,if any, effect on the peptide binding specificity. Likewise, suchresidues are not involved in TCR recognition. Alternatively,the existence of a rather extended humoral immune reactivearea downstream of the MHR that includes residue 213 (asrecently shown for residues 178 to 186) (30) or an eventual
crucial influence of such I213L replacement on the conforma-tional structure of the S protein should be further investigated.In any case, this and/or the remaining S gene changes couldaffect the S protein binding to anti-HBs antibodies, whichmight provide a putative explanation for the free cocirculationof both HBsAg and anti-HBs antibodies. Since the three-di-mensional structure of the HBV surface protein is still un-solved (3), the prediction of conformational, biochemical, andfunctional effects as a consequence of these amino acid sub-stitutions is merely speculative. Nevertheless, experiments in-volving site-directed mutagenesis and subsequent antigenicanalysis of all the amino acid changes herein reported are inprogress.
Bearing in mind the overlapping structure of the S and Pgenes of HBV (Fig. 1), amino acid substitutions within the Sgene might or might not account for amino acid substitutionsin the P gene and vice versa. In this study, analysis of theoverlapping fragment of HBV polymerase showed severalamino acid changes. One of the observed mutations (codon
FIG. 3. Mutated B- and T-cell-restricted epitopes derived from the S protein are shown. Pol-mutated epitopes (the overlapped region withinthe S protein is underlined) were observed within amino acids 455 to 463 (L463S in clone 1) and 504 to 512 (P509H in clone 1 and H508L in clone5) for MHC-I peptides and within amino acids 503 to 517 (P509H in clone 1 and H508L in clone 5) for the overlapped MHC-II peptide.
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489) resulted in the premature termination of the Pol protein(Table 1). It is assumed that since certain mutations in the Pgene may impair virus replication to a different extent, a minorpopulation of intact genomes should be present to help theformation of viral particles by complementation (38).
As expected for patients not treated with lamivudine, theactive site of the RT enzyme exhibited the wild-type aminoacid sequence (YMDD) (Fig. 1), where Met is usually replacedby either Val or Ile in lamivudine-resistant isolates.
In addition, it is suggested that the circulation of some of theobserved mutated HBV epitopes might represent a potentialpopulation risk, since neither current hepatitis B vaccines norhepatitis B immune globulin effectively prevents the liver dis-ease associated with some of them. Moreover, some of therecorded HBs antigen variants may influence the accuracy ofthe results obtained with currently used diagnostic tests, asreported with previously studied mutants (9, 12, 13, 19, 20, 30,37), since some of them may prove undetectable, althoughothers may not result in a complete loss of antigenicity (17).
In brief, this study underlines the unusual simultaneous de-tection of both humoral and cellular mutated HBV epitopesduring the natural course of a chronic HBV infection despitethe presence of anti-HBs antibodies. The extremely rareC107R replacement (reviewed in references 32 and 37) and theI213L unusual substitution (alone and combined with theherein reported exchanges) deserve to be further explored infuture in vitro studies of HBsAg–anti-HBs antibody interactions,since they may be immunogenic, although with a changed speci-ficity (42).
ACKNOWLEDGMENTS
We are indebted to A. M. Andreetta, J. Trinks, and Marıa de losAngeles Oubina for excellent technical assistance. We are truly grate-ful to Marıa Victoria Illas for enhancing readability.
This study was partly supported by the following grants: BID 1201/OC-AR-PICT 10.871 from the National Agency for Scientific andTechnological Promotion (ANPCyT), PIP 842 and PIP 6.065 from theNational Research Council (CONICET), and UBACYT M057 fromthe University of Buenos Aires.
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2198 CUESTAS ET AL. J. CLIN. MICROBIOL.
Disease progression due to dual infection in an HLA-B57-positive asymptomaticlong-term nonprogressor infected with a nef-defective HIV-1 strain
Martine Braibant a,⁎, Jing Xie b,c, Assia Samri b,c,d, Henri Agut e, Brigitte Autran b,c,f, Francis Barin a
a Université François-Rabelais, Inserm U966 & CHRU de Tours, 37032 Tours, Franceb Inserm, UMRS-945, Laboratoire d'Immunologie Cellulaire et Tissulaire, 75013 Paris, Francec UPMC Université Paris 06, UMRS-945, Laboratoire d'Immunologie Cellulaire et Tissulaire, 75013 Paris, Franced IFR113, Laboratoire d'Immunologie Cellulaire et Tissulaire, 75013 Paris, Francee Laboratoire de Virologie, UPRES EA2387, Hôpital Pitié-Salpêtrière, 75013 Paris, Francef AP-HP Hôpital Pitié-Salpêtrière, Laboratoire d'Immunologie Cellulaire et Tissulaire, 75013 Paris, France
a b s t r a c ta r t i c l e i n f o
Article history:Received 12 March 2010Returned to author for revision 7 April 2010Accepted 24 May 2010Available online 19 June 2010
We describe the case of an HLA-B57-positive long-term nonprogressor in whom we previously showed thatPBMCs accumulated HIV-1 subtype B proviruses defective in the env gene. After more than 10 years ofcontrol of infection, plasma viremia increased progressively, with a concomitant loss of CD4+ T cells. Byphylogenetic analyses of env, nef, vif, and gag sequences obtained at different time points, we suggest herethat this patient was initially infected by a putatively attenuated nef-defective variant and that loss of controlwas due to superinfection with a fully competent virus belonging to the same clade B. At the time ofsuperinfection, its plasma was unable to efficiently neutralize the superinfecting virus and moderate Gag-specific CD8+ T-cell responses were observed. This suggests the limited capacity of even a long-lastingnatural infection with a nef-deficient HIV-1 strain to elicit immune responses able to prevent and controlsuperinfection with a virus of the same clade.
Despite considerable efforts during the last two decades, allattempts to develop an effective human immunodeficiency virus(HIV) vaccine that prevents infection or significantly delays theprogression to AIDS have failed (Flynn et al., 2005; Gilbert et al., 2005a;Gilbert et al., 2005b; Steinbrook, 2007). Difficulties lie in propertiesspecific to HIV such as the particularly high sequence diversity incirculating isolates worldwide (Korber et al., 2001), the targeting ofcritical immune cells (Mehandru et al., 2004; Steinbrook, 2007) or themultiple strategies evolved by the virus to avoid immune elimination(Le Gall et al., 1998; Wyatt and Sodroski, 1998; Yang et al., 2002). Themost promising strategy for preventing HIV-1 infection would be avaccine that induces broadly neutralizing antibodies before infection(Barouch, 2008;Walker and Burton, 2008). Indeed, passive infusion ofbroadly cross-reactive neutralizing antibodies in animal modelseffectively confers protection against challenge infection (Baba et al.,2000; Ferrantelli et al., 2004; Hessell et al., 2009a; Hessell et al., 2009b;
Mascola et al., 1999;Mascola et al., 2000; Shibata et al., 1997). Vaccine-mediated protection from AIDS virus infection has only been achievedin primate models, using live-attenuated simian immunodeficiencyvirus (SIV) vaccines carrying major deletions in nef and/or otheraccessory genes (Almond et al., 1995; Daniel et al., 1992;Wyand et al.,1999; Wyand et al., 1996). Unfortunately, safety concerns havehampered the development of attenuated vaccines. Some animalsinfected with nef-deleted SIV progressed to AIDS in the absence ofwild-type challenge (Hofmann-Lehmann et al., 2003; Sawai et al.,2000). In addition, of the few described long-term nonprogressors(LTNP) naturally infected with nef-defective strains (Kirchhoff et al.,1995; Learmont et al., 1999), some showedCD4+T-cell loss aftermanyyears of asymptomatic infection (Birch et al., 2001; Churchill et al.,2004; Churchill et al., 2006; Gorry et al., 2007; Learmont et al., 1999).Nevertheless, considerable effort has been made to understand thefactors that promote protection against disease progression in suchpatients, to gain insight into protective immune responses, and thuspave the way for a protective HIV vaccine (Dyer et al., 1999;Greenough et al., 1994; Verity et al., 2007).
The LTNP status is not necessarily due to HIV infection with adefective virus. Host factors, such as the expression of protective humanleukocyte antigen (HLA) alleles, also appear to play an important role inthe control of viremia in some LTNPs infected by fully replicative HIV-1strains (Kaslow et al., 1996; Kiepiela et al., 2004; Lambotte et al., 2005).Major histocompatibility complex class I alleles, such as HLA-B57,
-B5801, and -B27 alleles, are associated with slower rates of HIV-1disease progression. Recent studies have shown that, in subjectsexpressing these alleles, the control of HIV-1 replication is linked to anHIV-1-specific CD8+ T-cell response directed, early in infection, againsta highly conserved regionwithin p24 Gag (Almeida et al., 2007; Streecket al., 2007). A nonprogressive clinical course in HLA-B57 and -B5801HIV-infected individuals has also been shown to be associated with apreserved CD8+ T-lymphocyte response to the HW9 epitope in Nef(Navis et al., 2008).
Here, we report an informative case of superinfection in a LTNPinitially infected with a nef-defective HIV-1 strain. Both initial andsuperinfecting viruses belonged to subtype B. Superinfection led toincreased viral replication and progressive loss of CD4+ T cells, despitethe expression of the protective HLA-B57 allele by this patient. Our datashowed the limited capacity of a natural infection with a putativelyattenuated HIV-1 strain to generate protective immune responses, bothin terms of induction of neutralizing antibodies and the generation andpreservation of specific cytotoxic T-lymphocyte responses.
Results
Loss of control of HIV-1 infection of patient 4050
Patient 4050, a 58-year-old homosexual male, was included in theFrench “Asymptomatiques à Long Terme” cohort (ANRS CO15) in June1995, after 10 years of seropositivity and 5 years of stable CD4+ T-cellcountN600 cells/mm3. He had no clinical symptoms and had notreceived antiretroviral therapy, thus fulfilling the LTNP criteria. Oninclusion in the cohort, he had extremely low viral loads, i.e., a plasmaHIV-1 RNA level of 135 copies/ml and a cellular HIV-1 DNA level of 28copies/106 PBMCs. However, after this long period of stability, plasmaviremia increased progressively, reaching 10,350 copies/ml in March1999 (month 45 following T1, the time of patient inclusion), with aconcomitant progressive decline in the CD4+ T-cell count to below 600cells/mm3 after January 1999 (Fig. 1). Patient 4050 initiated antire-troviral treatment in March 1999, a period when HAART was initiatedearlier than the current consensus, leading to the rapid control of viralreplication and an increase of CD4+ T-cell numbers.
Patient 4050 was infected initially by a nef-defective HIV-1 virus
We previously described sequences of the V1-to-V5 region of theenv gene obtained from the proviral DNA extracted from PBMCs ofpatient 4050, collected on inclusion in the cohort (Braibant et al.,
2008). In this previous study, analyses of 15 different clones revealeda high proportion of defective clones (14 of 15 clones, i.e., 93%) withnucleotide deletions leading to frameshifts and premature stopcodons in the env gene (Braibant et al., 2008). We hypothesizedthat the LTNP status of patient 4050, whose cells accumulatedproviruses defective in the env gene, may be associated with infectionby an HIV-1 isolate harboring deficient accessory proteins Vif or Nef,as reported by others (Calugi et al., 2006; Pace et al., 2006). Wetherefore amplified the complete vif and nef genes by nested PCRs andcloned the PCR products into the pCR2.1 vector. A total of 28 nef clonesand 30 vif clones present on inclusion were obtained and sequenced.Sequences analysis of vif clones, using HIV-1 NL4.3 as the referencesequence, did not show significant mutations (data not shown). Incontrast, a major genetic change was found in nef sequences. Allsequences contained a deletion of 20 nucleotides (between nucleo-tides 224 and 243 of the reference sequence), leading to a truncatedNef protein (premature stop codon at position 261 to 263 of thenucleotidic reference sequence) that lacked major functional motifs(for reviews, see Foster and Garcia, 2008; Geyer et al., 2001). Nucleicacid and amino acid consensus sequences derived from the quasis-pecies population in patient 4050 are shown in Fig. 2.
Loss of viral control was associated with superinfection
The loss of control of HIV-1 infection of patient 4050 after morethan 10 years of stability in presence of a partially defective genomeraised the possibility that this change could be due to HIV-1superinfection. To investigate this hypothesis, we compared env(V1–V5 region), nef, vif, and gag (part of the P24 coding sequence)sequences from PBMC samples collected at different time points. Foreach gene, 22 to 30 clones were obtained and sequenced at entry intothe cohort (T1) and 2 years later (T3). In addition, for gag and nefgenes only, 22 to 24 clones were obtained and sequenced at anintermediate time point, i.e., 1 year after entry into the cohort (T2).
Phylogenetic analyses of env, nef, vif, and, to a lesser extent, gagsequences clearly suggested the sequential infection of patient 4050by two phylogenetically distinct subtype B strains (strains 1 and 2)(Fig. 3). Vif, env, nef, and gag sequences were more similar toequivalent sequences of subtype B reference strains than to those ofstrains of other subtypes. All clones sequenced for nef, vif, and gag, andmost of those sequenced for env (22 of 25 clones), collected at T1,clustered in the same branch but were phylogenetically distinct fromthose recovered at T3, which clustered in another branch (Figs. 3A–D).The genetic distances between the two HIV-1 strains (strains 1 and 2)within the sequenced regions were 15.7%±1.3% in env, 4.0%±0.8% innef, 5.1%±0.9% in vif, and 2.9%±0.7% in gag. The large geneticdistance observed in env sequences provides further evidence of adual infection in patient 4050.
None of the env sequences recovered at T3 was genetically altered(Fig. 3A). However, only three of those recovered at T1 weregenetically intact. Two of these genetically intact sequences weremore closely related to those recovered at T3, and the third, althoughphylogenetically linked to sequences of strain 1, differed by a smallergenetic distance from env sequences of strain 2 (9.5%±0.8% versus theaverage genetic distance of 15.7%±1.3%) (Fig. 3A). The alignment ofthis third nucleotide sequence of envwith the consensus sequences ofstrain 1 and strain 2 suggested that a recombination event hadoccurred between these two infecting strains. This finding wasconfirmed using the recombinant identification program RIP 3.0(Fig. 4). The recombination breakpoint was found in the C2 constantregion of the envelope glycoprotein, in a previously described hotspotregion corresponding to the top portion of an RNA hairpin structure(Galetto et al., 2006; Galetto et al., 2004). These three genetically intactsequences suggested that strain 2 was already present in subject 4050at time of inclusion. However, the predominance of strain 2 after2 years showed that it is likely to have a growth advantage over strain
Fig. 1. Course of infection in patient 4050 over the 5 years after inclusion in the LTNPcohort. Plasma viral loads and CD4+ T cell counts are indicated by open diamonds andclosed squares, respectively. Time points at which blood samples were collected areindicated by arrows (T1, T2, and T3 : time at enrollment and 1 and 2 years later,respectively). The time at which treatment was initiated is indicated (HAART).
1 and suggested that the superinfection occurred at or not long beforeentry into the cohort.
All nef sequences determined at T1 (strain 1) were defective andphylogenetically distinct from those recovered at T3, which were notaltered (strain 2) (Fig. 3B). Twenty sequences recovered at T2clustered with strain 1 and two clustered with strain 2. However, incontrast to strain 1 sequences,most T2 sequences (18 of 20) clusteringwith strain 1 were not altered. The alignments indicated that theyresulted from a recombination between the two infecting strains, asshown using the recombinant identification program RIP 3.0 (Fig. 5).
The majority of vif and gag sequences were not altered, whetherfrom strain 1 or strain 2 (Fig. 3C). The sequenced region of gag waschosen as it contained the most common CTL HLA-B57-restrictedepitopes. However, this regionwas short (448 nucleotides) and highlyconserved, even among different strains of subtype B. Thus, theclustering of sequences, and whether they belonged to a singleevolving strain or to two different strains, was not so clear (Fig. 3D).Nevertheless, sequences recovered at T1 were phylogeneticallydistinct from those at T3 and, although the genetic distances betweenthe two populations and the bootstrap values were smaller than for
Fig. 2. Alignment of nucleic acid and deduced amino acid nef sequences. A, Nucleic acid consensus sequence derived from the quasispecies population recovered from DNA extractedfrom PBMCs of patient 4050, collected at time of inclusion in the cohort, is aligned with the nef sequence of the NL4.3 reference strain. Identical and absent nucleotides arerepresented by dots and dashes, respectively. The observed 20 nucleotides deletion and the resulting premature stop codon are highlighted in gray. B, Amino acid consensussequence is aligned with NL4.3 Nef sequence. Identical and absent nucleotides are represented by dots and dashes, respectively. Known Nef functional motifs/regions are indicated.Regions playing a role in CD4 andMHC-1 down-regulation are indicated by CD4↓ andMHC-1↓. These regions include motifs involved in the internalization and trafficking of Nef [i.e.,a WL motif interacting with the cytoplasmic tail of CD4, an acidic cluster of four glutamic acids (EEEE) interacting with PACS-1, a highly exposed cluster of three amino acids (FDP)interacting with the human thioesterase, a di-glutamic acid (EE) motif interacting with β-COP in endosomes, a di-leucine (LL) motif interacting with adaptor complexes AP-1 andAP-2, and a di-aspartic acid motif (DD) interacting with the subunit H of the vacuolar ATPase (V1H)], motifs involved in cellular activation and signaling by Nef [i.e., a proline-richmotif (PxxPxxPxRP) interacting with the SH3 domains of Src family kinases and two arginine residues (RR) interacting with members of the p21-activated kinase (Pak) family] andone motif involved in Nef modification [the N-myristoylation site (MGxxxS/T)].
the other genes, we could speculate by analogy that they belong totwo different strains (strain 1 and strain 2) (Fig. 3D). Almost all thesequences recovered at T2 (23 of 24), although forming a clusterdistinct from the T1 sequence cluster, were more closely related to T1than to T3 sequences (average genetic distances of 1.6%±0.05%for sequences found at T1 versus 2.7%±0.07% for those found at T3).We therefore speculated that they belong to the same initial strain(strain 1) and resulted from a series of genetic changes in this strain,over the course of one year. By contrast, one sequence recovered at T2clustered with sequences recovered at T3, and was therefore probablyderived from strain 2.
Absence of heterologous neutralizing antibody responses
We previously assessed the ability of plasma from patient 4050,collected at time of inclusion in the cohort, to neutralize a panel offour heterologous primary isolates belonging to four different cladesor circulating recombinant forms (B, F, CRF01-AE, CRF02-AG)(Braibant et al., 2008). Plasma from patient 4050 did not displayneutralizing activity against any of the four strains tested, even at thelower plasma dilution used. We carried out more specific tests todetermine whether plasma from patient 4050 contained neutralizingantibodies against the superinfecting virus on entry into the cohort.We thus studied the capacity to neutralize pseudoviruses expressingfunctional envelope proteins generated using five env sequencesrecovered from the superinfecting virus, i.e., 1 of the 2 clonedsequences (CL6) recovered at time of entry into the cohort and 4 ofthe 22 sequences (CL1 to CL4) obtained 2 years later (Fig. 3A). Theneutralization profiles for each of the five pseudoviruses were verysimilar. Neutralization levels were very low, failing to reach 90%neutralization even using a 1:20 dilution of plasma, the highestplasma concentration tested (Fig. 6). The calculated IC50 values(defined as the reciprocal of the plasma dilution resulting in 50%neutralization) were very low, ranging from 125 to 167. We furtherevaluated the presence of neutralizing antibodies in this patient'splasma by testing the ability to neutralize pseudoviruses expressingthe env sequence cloned from the laboratory adapted strain NL4-3 ofsubtype B, which is known to be sensitive to neutralization.Neutralization of these pseudoviruses reached 90% using the twohighest concentrations (1:20 and 1:60) (Fig. 6). Calculated IC90 andIC50 values were 167 and 941, respectively.
Time course of CD8+ cellular immune responses
To address the importance of HIV-specific CTL responses generatedby infection with the nef-defective strain and subsequently by thesuperinfecting strain, we tested PBMCs collected at T1, T2, and T3 forresponses against pools of various synthetic 15-mer peptides, using anIFN-γ-ELISpot assay. These peptides spanned the entire HIV-1-Gag(three p17 pools, five p24 pools, and three PP pools), HIV-1-RT (fourpools), and HIV-1-Nef (three pools) sequences of HxB2. At T1, whenthe initial nef-defective HIV-1 strain predominated, only two p24peptide pools were recognized by CD8+ T cells (p24 pools 1 and 3;350 and 325 SFC/106 PBMCs, respectively) (Figs. 7A–C). At T2, whenthe superinfecting virus was present but not yet predominant, CTLresponses increased. A total of nine peptides pools, including one poolof p17, four pools of p24, one pool of PP, one pool of RT, and two poolsof Nef, were recognized by CD8+ T cells. Again, the two mainlyrecognized pools were p24 peptide pools 1 and 3 (777 and 550 SFC/106 PBMCs, respectively) and, to a lesser extent, p24 peptide pool 4(210 SFC/106 PBMCs), and Nef peptide pool 3 (310 SFC/106 PBMCs).
At T3, when the superinfecting virus predominated, the CTL responseswere weaker and were similar to those detected at T1 (p24 pools 1and 3; 297 and 270 SFC/106 PBMCs, respectively).
The two most frequently recognized p24 pools (pools 1 and 3)cover amino acids (aa) 133 to 187 and 221 to 275 of Gag, respectively,and therefore include the HLA-B57-restricted IW9, KF11 and TW10epitopes. The Nef peptide pool 3 covers aa 97 to 147 and includes theHLA-B57-restricted HW9 epitope. To determine whether the ob-served responses were targeted to these HLA-B57-restricted epitopes,peptides corresponding to these epitopes were tested individually. Inaddition, one peptide corresponding to the HLA-B57-restricted YY9Nef epitope (not included in any used Nef peptide pools) was alsotested. Due to the lack of PBMCs collected at T3, this was carried out attwo time points only (T1 and T2).
For Gag, the two main targeted epitopes were indeed IW9 (230 and895 SFC/106 PBMCs at T1 and T2, respectively) and KF11 (405 and 540SFC/106 PBMCs at T1 andT2, respectively). By contrast, the TW10epitope,the early and immunodominant targeting ofwhich has been correlated tothe nonprogression of HIV-1 infection inHLA-B57 individuals, gave rise tomore moderate responses (105 and 240 SFC/106 PBMCs at T1 and T2,respectively). Analysis of Gag viral sequences revealed the presence of apreviously describedTW10CTL escapemutation (T242N) in all sequencesrecovered fromthe superinfecting strain, i.e., all sequences recoveredat T3and the single related sequence recovered at T2 (Figs. 3D, 7, and 8D).Analysis of CTL responses to the mutated epitope TW10mut revealed aweaker response to the mutated epitope at T2 (155 and 240 SFC/106
PBMCs for TW10mut and TW10 wild type, respectively), suggesting thatthe lowCTL response to this keyepitopemayhave impaired theearly viralcontainment of the superinfecting strain.
For Nef, neither wild-type HW9 nor wild-type YY9 was recognizedat any time. Analysis of viral sequences showed one mutation in HW9(HW9mut F121L) in 3 of the 22 sequences recovered at T2 and in all 25nondefective sequences recovered at T3 and one mutation in YY9(YY9mut1 Y135F) in all sequences recovered at T2 and T3 (Figs. 3B, 5,and 8D). An additional mutation in YY9 (YY9mut2 I133T)was observedfor most of the sequences recovered at T2 but was no longer observedat T3. No CTL responses against these mutants were observed except aweak response against HW9mut at T2 (105 SFC/106 PBMCs).
Discussion
We describe an HLA-B57-positive LTNP (patient 4050), in whomwe previously showed that PBMCs accumulated HIV-1 subtype Bproviruses with a defective env gene (Braibant et al., 2008). However,after more than 10 years of control of infection, plasma viremiaincreased progressively, with a concomitant progressive loss of CD4+
T cells. Here, the data suggest that this patient was initially infected bya nef-defective HIV-1 variant and that loss of control was due tosuperinfection with a fully competent virus belonging to the sameclade B. Although we could not precisely determine the time ofsuperinfection due to the lack of available samples before enrollment,the recovery at T1 (at entry in the cohort, after N10 years of infection)of two env sequences related to those of the superinfecting strainsuggested that this patient was already superinfected at that time.However, the few env sequences recovered and the complete absenceof nef, gag, and vif sequences belonging to the superinfecting strain atT1 suggested that superinfection occurred at or not long before T1,and at least probably not around the time of primary infection. Thisassumption is strengthened by the extremely low cellular viral loadaccumulated at T1 (28 copies/106 PBMCs), in accordance withinfection by a poorly replicative HIV-1 strain.
Fig. 3. Phylogenetic analysis of env (A), nef (B), vif (C), and gag (D) sequences obtained from PBMC samples collected at time of inclusion (T1, closed triangles), 1 year after inclusion(T2, gray diamonds) and 2 years after inclusion (T3, open circles). A distance scale is given for each neighbor-joining tree. Bootstrap values are expressed as percentages per 500replicates; only values above 60% are indicated on nodes. Reference sequences of various subtypes are included in each tree. Each of these sequences is indicated by its subtype andnucleotide accession number. Defective sequences are indicated by asterisks.
Accumulation of cells containing env-defective proviruses waspreviously reported in an LTNP infected with nef-deficient virus(Calugi et al., 2006). A similar observation made in our patient mightsuggest that nef and env deletions might be linked. As previously hy-pothesized, it seems likely that, in the absence of down-regulationof class 1 major histocompatibility complex molecules by Nef, CD8+
cytolysis is more efficient in eliminating cells presenting wild-type Env,whereas cells with truncated Env accumulate (Calugi et al., 2006).Althoughwedid not analyze the functional properties of theNef proteinin the nef-defective HIV-1 variant infecting our patient, we can predictthat, expressed or not, its truncated form lackingmajor domains wouldnot be functional. The 20 nucleotides deletion observed in nef clones didnot contain any B57-restricted epitopes and therefore could not be aresponse to a selective pressure from CTL responses.
Superinfection of patient 4050 showed that the nef-deficient HIV-1strain was unable to induce immune responses that protect againstHIV-1 superinfection or that control the new virus after superinfec-tion. This raised the question of the extent of neutralizing antibodiesand/or CTL responses generated by such a putatively attenuated HIV-1 strain. A number of studies have shown that broadly neutralizingantibody responses are more frequent in LTNP than in other HIV-1-infected patients (Cao et al., 1995; Carotenuto et al., 1998; Cecilia etal., 1999; Pilgrim et al., 1997). However, none of these studies ad-dressed the question taking into account the properties of the genes—intact or delete—of the infecting viruses. Several reports have shownthat nef-deleted SIV may induce neutralizing antibodies in macaques(Cranage et al., 1997; Enose et al., 2002; Kumar et al., 2002; Montefioriet al., 1996; Stipp et al., 2000). A recent study of the antibodyresponses in eight blood transfusion recipients infected with a nef-attenuated HIV-1 acquired from a single donor (Sydney Blood BankCohort) showed a strong association of broader neutralizing antibodyresponses with higher viral load and a higher risk of diseaseprogression (Verity et al., 2007). This highlights the importance ofviral replication in mounting a robust humoral immune response, aswe observed in a previous study (Braibant et al., 2006). Three LTNPs inthis cohort, who had remained asymptomatic with undetectableplasma viral load and stable CD4+ T-cell counts since 1993, had littleor no neutralizing activity in their sera. In the case of patient 4050 inthis study, we found that, at the time of superinfection, his plasma didnot contain neutralizing antibodies against four heterologous primaryisolates, including one subtype B strain (Braibant et al., 2008), butneutralized the neutralization-sensitive NL4-3 strain. Using an Env-pseudotyped virus neutralization assay, we also showed that thepatient's serum did not contain antibodies able to efficientlyneutralize the superinfecting virus. Although we cannot be sure thatthe presence of heterologous neutralizing antibodies would have beenefficient in preventing superinfection if they had been present, itseems likely that the risk of superinfection was increased by the lackof such antibodies. The control of HIV-1 in HLA-B57 patients has beenattributed to an early and immunodominant CTL response targeting ahighly conserved Gag epitope, TW10 (Streeck et al., 2007). In patient4050, dominant responses were observed at T1 and one year aftersuperinfection and were directed mainly toward two epitopes in Gag,IW9 and KF11, but more moderately toward TW10. The two majortargeted epitopes, IW9 and KF11, were conserved in the uncontrolledsuperinfecting virus suggesting that these specific CTL responses wereinefficient at controlling infection by a fully competent virus.However, we cannot exclude the possibility that these responses
contributed to the control of the nef-defective variant expressinghigher levels of major histocompatibility class I (MHC-I) molecules.The down-regulation of MHC-I molecules by Nef is a highlydocumented potential mechanism developed by the virus to bluntcytotoxic T-cell recognition of infected cells (Malim and Emerman,2008). In addition, the diversity of functional properties assigned toNef, i.e. CD4 down-regulation, enhancement of virion infectivity,T-cell receptor (TCR-CD3) regulation (Malim and Emerman, 2008),might have contributed to the control of the replication of the nef-deleted virus.
In contrast, the superinfecting virus had a T242N mutation in theTW10 epitope, previously described as an escape mutation fromHLA-B57 restricted cytotoxic T cells that causes a fitness cost for thevirus (Martinez-Picado et al., 2006). The even lower CTL response tothis mutated epitope compared to that toward the wild type TW10epitope may suggest that the occurrence of this mutation contributedto the lack of control of the incoming virus. Nevertheless, as we didnot determine the sequence of the donor virus, we cannot exclude thepresence of this mutation already at the time of transmission. Despitethe previously described fitness cost for the virus, we showed here inpatient 4050 that the virus carrying this mutationwas associated witha progressive disease course. This could be attributed to compensa-tory mutation(s) that restore the replication capacity of the mutant.However, none of the previously described upstream mutations inGag shown to partially restore replication of the T242N variant werefound in sequenced Gag clones of the superinfecting virus (Brockmanet al., 2007; Kirchhoff et al., 1995; Martinez-Picado et al., 2006). Insummary, although a specific CTL response against Gag was present inthe patient at T1, including two HLA-B57-restricted epitopes, thisresponse was not sufficient to control the superinfection. No Nef-specific CTL response was present at T1, an observation that seemslogical due to exposure only to the Nef-deficient virus at that time.
The reported case brings additional elements suggesting that thepresence of weak neutralizing antibodies and/or specific CTLresponses are inefficient for elimination of the virus or a successfulcontainment preventing the development of the disease whennaturally exposed to a fully competent HIV strain. This highlightsthe considerable difficulties that remain to generate immunogens ableto inducemore potent immune responses than those observed in HIV-1-infected patients with long-lasting infection.
Conclusions
Overall, our study suggests the limited capacity of even a long-lasting natural infection with a putatively attenuated nef-deficientHIV-1 strain to elicit potent immune responses able to prevent andcontrol superinfection with a second virus of the same clade. Suchobservations in this and other reports of superinfections (Altfeld et al.,2002; Blish et al., 2008; Casado et al., 2007; Jost et al., 2002; Streecket al., 2008) highlight the considerable difficulties that need to beovercome to develop a successful rational for a preventive vaccine.
Materials and methods
Study subject
Patient 4050 was recruited to the French “Asymptomatiques à LongTerme” cohort (ALT ANRS CO15) in 1995. The ALT status was defined as
Fig. 4. Recombination between strain 1 and strain 2 in env sequences. A, Recombinant nucleic acid env sequence is aligned with consensus strain 1 and strain 2 env sequences.Identical and absent nucleotides are represented by dots and dashes, respectively. When different from the recombinant env sequence, consensus sequence nucleotides arerepresented by uppercase letters if conserved in 100% of sequences and by lowercase if conserved in more than 50% of sequences. Env variable regions are highlighted in gray. Theregion presumed to contain the recombination breakpoint is outlined by a box. B, Graph generated using the Recombinant Identification Program—RIP 3.0. Red and green curvestrace the similarity between the recombinant env sequence and the consensus strain 1 and strain 2 sequences, respectively. Each plotted point represents the distance value at thecenter of a window (size of 200) moving in increments of one nucleotide residue from left to right in the alignment. The lower horizontal colored bar represents the best matchedsequence. The upper horizontal colored line shows whether the best match is significantly better than the second match.
87M. Braibant et al. / Virology 405 (2010) 81–92
an asymptomatic HIV-1 infection for at least 8 yearswith stable CD4+ T-cell countN600 cells/mm3 over the previous five years and noantiretroviral therapy (Candotti et al., 1999; Magierowska et al., 1999;Martinez et al., 2005; Ngo-Giang-Huong et al., 2001). Patient 4050 was
infected with a subtype B virus. Plasma viral load and DNA viral loadwere determined as previously described (Candotti et al., 1999;Rouzioux et al., 2005). Blood samples were collected at each visit,every year, as described in the follow-up protocol for this cohort.
Fig. 5. Recombination between strain 1 and strain 2 in nef sequences. A, Recombinant nucleic acid nef sequence is alignedwith consensus strain 1 and strain 2 nef sequences. Identicaland absent nucleotides are represented by dots and dashes, respectively. When different from the recombinant nef sequence, consensus sequence nucleotides are represented byuppercase letters if conserved in 100% of sequences, and by lowercase if conserved in more than 50% of sequences. The presumed recombination breakpoints are shown in boxes.Nucleotides encoding B57-restricted epitopes in recombinant and consensus strain 2 sequences are highlighted in grey. Amino acids deduced sequences are represented above andbelow recombinant and consensus strain 2 sequences, respectively. B, Graph generated using the Recombinant Identification Program -RIP 3.0. Red and green curves trace thesimilarity between the recombinant nef sequence and the consensus strain 1 and strain 2 sequences, respectively. Each plotted point represents the distance value at the center of awindow (size of 100) moving in increments of one nucleotide residue from left to right in the alignment. The lower horizontal colored bar represents the best matched sequence. Theupper horizontal colored line shows whether the best match is significantly better than the second match.
Genomic DNA was extracted from about 5 million frozen PBMCscollected at each time point, using the QIAamp DNA Blood Midi kit(Qiagen). Separate nested PCRs were used to amplify four regions of theHIV-1 genome, consisting of a 1.2-kb fragment of the env geneencompassing most of the gp120 coding sequence (from upstream V1to downstream V5, nt 5941 to 7216 in the HXB2 genome), the completenefgene (nt 8343 to 8714 in theHXB2genome), the complete vifgene(nt4587 to 5165 in the HXB2 genome), and a fragment of the gag geneencompassing part of the P24 coding sequence (nt 717 to 1165 in theNL4.3 genome). Subtype B env-specific primers and conditions of envamplificationwerepreviouslydescribed (Braibant et al., 2008). SubtypeB
nef-specific outer primerswere nefB-sext (5′-GCAGTAGCTGAGGGGACAG-ATAGGGTTATAG-3′) andnefB-asext (5′- CTCCCAGTCCCGCCCAGGCCAC-3′),and inner primers were nefB-sint (5′- GACAGGGCTTGGAAAGGG-CTTTGCTATAA-3′) and nefB-asint (5′- TCAGCAGTTCTTGTAGTACTCC-GGATGC-3′). Subtype B vif-specific outer primers were vifB-sext(5′-AGATAATAGTGACATAAAAGTAGTRCCAAG-3′) and vifB-asext (5′-AAGTATCCCCATAAGTTTCATAGATATRTTG-3′), and inner primers werevifB-sint (5′- GTRCCAAGAAGAAAAGCAAAGATCATTAGRG-3′) and vifB-asint (5′- CCTAAGCCATGGAGCCAAATCCTAGGAAART-3′). SubtypeB gag-specific outer primers were gagB-sext (5′-CAAAAGTAAGAAAAARGCA-CARC-3′) and gagB-asext (5′- CCAGAATGCTGGTAGGRCTRTA-3′), andinner primers were gagB-sint (5′- GGTCAGCCAAAATTACCCHATA-3′)and gagB-asint (5′- CATYCTTACTATTTTATTTAATCCYA-3′). Amplifica-tions were carried out with the Platinum PCR SuperMix High Fidelity(Invitrogen) under the following cycling conditions: 5 min at 94 °C,followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C for vif and nef or 30 sat 50 °C for gag and 1 min at 68 °C, and a final extension step of 7 min at68 °C. A 5-µl aliquot of the products of the first round of PCR was thentransferred to a new reaction mixture containing the inner primer pair,and a second round of amplification was performed under the samecycling conditions. All PCR products were inserted into pCR2.1 (Topo TAcloning kit; Invitrogen), according to the manufacturer's instructions.
Sequence analysis
All pCR2.1 clones were sequenced according to the Dye Terminatorcycle sequencing protocol (Applied Biosystems, Foster City, Calif.).Nucleotide sequences were assembled with the BioEdit package(version 5.0.9) and aligned using Clustal W (Hall, 1999; Thompson etal., 1994). Phylogenetic analysis and tree reconstructions wereperformed by the neighbor-joining method, with MEGA version 3.1(Kumar et al., 2004; Saitou and Nei, 1987). The distance matrix wascalculated with the two-parameter Kimura algorithm (transition-to-transversion ratio of 2.0) (Kimura, 1980). Approximate confidencelimits for individual branches were assigned by bootstrap resamplingwith 500 replicates.
Fig. 6. Absence of potent neutralizing antibodies against the superinfecting strain.Neutralization activity in plasma from patient 4050 collected at time of entry into thecohort was determined for viruses pseudotyped with functional envelope proteinscorresponding to the 5 env sequences from the superinfecting virus (CL1 to CL4 andCL6) or with the envelope protein of the laboratory adapted strain NL4-3. Percentage ofneutralization is plotted as a function of serum dilution.
Fig. 7. Alignment of consensus strain 1 and strain 2 gag sequences. Identical nucleotides are represented by dots. Consensus sequence nucleotides represented by uppercase lettersare conserved in 100% of sequences; those in lowercase are conserved in more than 50% of sequences. Nucleotides encoding B57-restricted epitopes are highlighted in grey andamino acids deduced sequences are represented above and below consensus strain 1 and strain 2 sequences, respectively. Numbering is that of the complete gag HxB2 referencesequence.
Subcloning of gp120 env gene clones into expression plasmid
The complete gp160 NL4-3 env gene was inserted into the EcoRIsite of the pCR2.1 vector.
Part of the env gene coding for most of the gp120 was digested outof this construct using NdeI and MfeI (New England Biolabs) andreplaced by the corresponding gp120 sequences excised from thepCR2.1 vector by digestion with the same enzymes. Chimeric envgenes were then cloned into the EcoRI site of the pCI expression vector(Promega).
Env-pseudotyped virus neutralization assay
Env-pseudotyped viruses were generated by cotransfecting 3×106
293T cellswith 4 μg of each pCI-envplasmid and8 μg of pNL4.3.LUC.R-E-(Connor et al., 1995), using FuGene-6 HD Transfection Reagent (RocheApplied Biosciences). After titration, pseudotyped virus stocks werediluted to obtain 1000 TCID50/ml in growth medium. Aliquots of 50 μl,corresponding to 50 TCID50, were then incubated for 1 h at 37 °C with50 μl of three-fold serial dilutions of heat-inactivated serum. The virus/
serummixture was then used to infect 10,000 TZM-bl cells (Platt et al.,1998; Wei et al., 2002) in the presence of 30 μg/ml DEAE-dextran.Infection levels were determined after 48 h, using the Bright-Gloluciferase assay (Promega) and a Centro LB 960 luminometer (BertholdTechnologies) to measure luciferase activity in cell lysates. The assaywas performed in duplicate and results were expressed asmean values.Neutralizing antibody titers were defined as the reciprocal of the serumdilution required to reduce relative luminescence units (RLUs) by 50%.
CD8+ T-cell epitopes and IFN-γ-ELISpot assay
Synthetic 15-mer peptides, overlapping by 11 amino acids (Neosys-tem, Strasbourg, France, kindly supplied by ANRS) and spanning the HIV-HxB2Gag, RT, andNef sequences,were divided into 18pools: 3 p17pools,5 p24 pools, 3 PP (i.e., p2/p7/p1/p6) pools, 4 RT pools, and 3 Nef pools.Nine CD8+ T-cell epitopes corresponding to HIV- Gag or Nef proteinsequences were synthesized: ISPRTLNAW (Gag147–155, IW9), KAFSPE-VIPMF (Gag162–172, KF11), TSTLQEQIGW (Gag240–249, TW10),TSNLQEQIGW(TW10mut: T242N), HTQGYFPDW (Nef116–124, HW9),HTQGYLPDW (HW9mut: F121L), YTPGPGIRY (Nef127–135, YY9),
Fig. 8. Time course for CD8+ cytotoxic T-lymphocytes responses. Total CD8+ T-cell responses specific for the Gag protein (A), the RT protein (B), and the Nef protein (C) wereassessed at three time points (T1, T2, and T3) in IFN-γ-ELISpot assays by stimulation with overlapping peptide pools (three p17 pools, five p24 pools, and three PP pools for Gag, fourpools for the RT and three pools for Nef). Sequences of known HLA-B57 restricted epitopes in Gag (IW9, KF11, and TW10) and Nef (HW9 and YY9) and of their mutated variants(TW10mut, HW9mut, YY9mut1, and YY9mut2) observed in viral sequences recovered from patient 4050 are shown in panel D. The number of viral sequences corresponding to each ofthese wild-type or mutated epitopes is shown. Peptide-specific CD8+ T-cell responses were tested individually for Gag and Nef epitopes (panels E and F, respectively) at two timepoints (T1 and T2).
YTPGPGIRF (YY9mut1: Y135F), and YTPGPGTRF (YY9mut2: I133T, Y135F).All peptides were more than 80% pure, according to HPLC profiles. TheIFN-γ-ELISpot assays were performed as previously described (Martinezet al., 2005). PBMC samples (viability above 85%) were stimulated withGag, RT, and Nef peptide pools (2 μg/ml for each peptide) or individualpeptides for18hours. ThecontrolsweremediumaloneorPHA(0.5 μg/ml,Abbott, Rungis, France). Spots were counted with an ELISpot reader(Axioplan-2 imaging, Zeiss, Germany). Data were expressed as SFC/106
PBMCs and considered positive if above 50 SFC/106 PBMCs aftersubtracting mean background.
Nucleotide sequence accession numbers
All sequences have been submitted to GenBank and assignedaccession numbersEF179866–EF179880 and GU457024–GU457261.
Acknowledgments
This work was supported by funding from the Agence Nationale deRecherches sur le Sida (ANRS, Paris, France) and Sidaction (Paris,France).
We thank all patients and clinicians (particularly Olga Kalmykova,Severine Deplat and Bertrand Dupond) who participated in the«A-symptomatiques à Long Terme»ANRS cohort (ANRS CO15).
pNL4.3.LUC.R-E- was obtained through the NIH AIDS Research andReference Reagent Program, Division of AIDS, NIAID, NIH: pNL4.3.LUC.R-E- from Dr. Nathaniel Landau.
The TZM-bl cells were obtained through the NIH AIDS Researchand Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-blfrom Dr. John C. Kappes, Dr. Xiaoyun WU and Tranzyme Inc.
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