Immune tolerance induction: What have we learned over time?

Development of inhibitory antibodies to infused factor VIII (FVIII) concentrates continues to be the most serious complication of haemophilia A management. Induction of immune tolerance by administering high doses of FVIII concentrate (antigen) and prothrombin complex concentrates to control bleeding was originated in the 1970s in Bonn, Germany, by Dr Hans-Hermann Brackmann, and became known as the Bonn protocol. ITI transformed the life of the index patient, who was 19 years of age when he began treatment, and dramatically improved the medical landscape for all patients with haemophilia and inhibitors. Over the past 40 years, variations to the Bonn protocol have been proposed. All protocols are effective although some are better suited than others for use in certain situations. The specific molecular defect in FVIII and the human leucocyte antigen (HLA) type of an individual with haemophilia are major codependent determinants to inhibitor development. Given the range of potential molecular defects and the staggering number of potential HLA types, it is likely that treatment arms of randomized studies in haemophilia represent highly diverse populations, which reduces the power of a study to demonstrate differences between treatments. Although available clinical guidelines and consensus recommendations for ITI therapy are not always in complete agreement, collectively the guidelines provide a reasonable level of guidance for administering ITI therapy under different clinical scenarios. Several studies of ITI therapy are ongoing with the aim of clarifying unresolved issues in haemophilia management including the role of von Willebrand factor in inhibitor eradication.

[Comprehensive Care Center Bonn from 1980 to 2009. Changes in the epidemiology and regional composition of the haemophilia population]. / Hämophilie-Zentrum Bonn 1980-2009. Wandel der Epidemiologie und der regionalen Zusammensetzung der Hämophilie-Population.

The Bonn Haemophilia Care Center provides patient care on a superregional level. The centre's large service area is, in part, due to the introduction of haemophilia home treatment and related to this the individualized prophylaxis in children and adults by Egli and Brackmann in Bonn in the early 1970s, that represented a milestone in German haemophilia therapy. Epidemiologic patient data from the two selected time points, 1980 and 2009, are evaluated to illustrate the change in the composition of the patient clientele. In 1980 a total of 639 patients were treated at the Bonn Haemophilia Center. 529 patients exhibited a severe form and 110 a non-severe form of the respective clotting disorder. In 2009 the Bonn Haemophilia Center took care for a total of 837 patients. There were 445 patients who suffered from a severe form of the considered clotting disorder while 392 showed a non-severe course. The number of less severely affected patients has increased significantly in 2009. Patients in 1980 were predominantly suffering from a severe form and most had to travel more than 150 km from their homes to the treatment center. In 2009 the number of patients living a medium-long distance from the care provider has significantly increased while the number of patients living more than 150 km from the center has decreased. Comparing 2009 to 1980 a growth of the center's regional character becomes apparent, especially when patient age and severity of the coagulation disorder are taken into consideration. The regional character was more strongly pronounced with milder disease severity and lower patient age. Due to the existence of well established primary haemophilia care in CCCs in Germany, the trend for the recent years is that the proportion of young patients that choose haemophilia care providers closer to their homes is increasing.

HLA genotype in patients with acquired haemophilia A.

Acquired haemophilia A (AH) is a rare bleeding disorder caused by an auto-antibody to coagulation factor VIII. It is associated with various autoimmune diseases, pregnancy, cancer or drug ingestion; however, in 50% of patients, no underlying disorder is found. In the present study, we investigated the association of HLA class I (A, B and Cw) and class II (DRB1 and DQB1) alleles with AH in a cohort of 57 patients. While no association with any class I allele was detected, a significantly higher frequency of DRB1*16 [odds ratio (OR) 10.2, 95%CI: 5.32-19.57, P < 0.0001] and DQB1*0502 (OR 2.2, 95%CI: 1.12-4.54, P < 0.05) was observed. In contrast, the frequency of DRB1*15 and DQB1*0602 alleles was found to be decreased in patients with AH corresponding to an OR of 0.4 for both HLA loci. Upon comparing the frequencies of these alleles with those of patients with congenital haemophilia A with inhibitors, the data demonstrate that the high risk alleles in patients with AH DRB1*16 and DQB1*0502 are found to be low risk alleles in patients with congenital haemophilia A with inhibitors (OR 1.1 and 1.5 respectively). Conversely, the alleles that exhibit low risk in AH DRB1*15 and DQB1*0602 are found to be high risk for haemophilia A inhibitor patients (OR 2.2 and 3.7 respectively). The pathophysiological reason for this finding remains unknown. It might be speculated that the presence or absence of the FVIII antigen and the various ability of HLA molecules to present the FVIII antigen to the T-cell receptor contribute to these findings.

The relevance of the bleeding severity in the treatment of acquired haemophilia - an update of a single-centre experience with 67 patients.

Acquired haemophilia (AH), an autoimmune disorder with clinical features ranging from harmless haematomas to life-threatening bleedings, still has a mortality rate of up to 25%. Owing to its low frequency (1-4 x 10(6)), standardized treatment protocols for its variable manifestations are not available. In case of prominent severe bleedings, the treatment should aim at rapid elimination of the antibody to protect patients from bleedings and on reinduction of long-term immune tolerance. Clinical data, short- and long-term treatment results of 67 patients diagnosed by our centre are presented. Patients were treated depending on their bleeding severity either by an immunosuppressive treatment alone, or in case of life-threatening bleedings, by a combined protocol (modified Bonn-Malmö protocol, MBMP) consisting of antibody depletion through immunoadsorption, intravenous immunoglobulin treatment, immunosuppression and high-dose factor VIII (FVIII) substitution. Mild bleedings occurred in two patients who were treated successfully alone by immunosuppression. Complete remission (CR) was achieved in 90% of the patients treated with MBMP (60). Of the six patients (10%) who achieved a partial remission (PR), four suffered from cancer. Mortality under MBMP was not seen. In contrast, five patients, in whom diagnosis of AH was delayed, experienced fatal outcome during surgical interventions before initiation of MBMP treatment. Prognosis in AH depends mainly on its prompt diagnosis. Treatment procedures should be adapted to bleeding severity and inhibitor titres. Under these conditions, AH is a potentially curable autoimmune disorder with an excellent prognosis.

Frequency of contamination of coagulation factor concentrates with novel human parvovirus PARV4.

Human parvovirus, PARV4 was identified in a plasma sample from a patient presenting with symptoms resembling acute HIV infection. Further strains of PARV4 and those of a closely related variant virus, were identified in plasma pools used in the manufacture of blood derivatives. DNA sequence analysis of these strains demonstrated two distinct PARV4 genotypes. It has subsequently been proposed that transmission of PARV4 occurs by parenteral routes. To investigate the risk of contamination of plasma-derived coagulation factor concentrates, we analysed 169 lots for PARV4 DNA by polymerase chain reaction. Positive samples were confirmed by nucleotide sequence analysis and quantification of the viral load. Twenty-one lots, representing eight different products were administered until the beginning of the 1980s and were not virally inactivated. Two lots examined were used in 1997, and 146 lots representing 13 products had been administered between October 2000 and February 2003. PARV4 DNA was detected in 7(33%) of the formerly administered lots, in one lot used in 1997, and in 13(9%) recently used lots. PARV4 genotype 2 DNA was predominantly present in the older concentrates, whilst genotype 1 was found more frequently in recently used lots. In three lots, both PARV4 genotypes were detected. Viral loads ranged between <100 and 10(5.8) copies mL(-1) of product, with higher viral loads in the older concentrates. The results show that PARV4 contamination can be detected in an appreciable proportion of clotting factor concentrates. Further studies are needed to determine whether or not PARV4 contamination of coagulation factors causes harm to the product recipients.

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

Protection against parenteral HIV-1 infection by homozygous deletion in the C-C chemokine receptor 5 gene.

OBJECTIVES: To investigate the role of the CC chemokine receptor 5 (CCR5) for parenteral transmission of HIV-1. DESIGN: The prevalence of the delta32 deletion within the CCR5 gene was determined in a cohort of 207 patients, who had received documented amounts of non-antibody-tested and non-inactivated clotting factor concentrate. METHODS: Chromosomal DNA of haemophiliacs was isolated from whole blood. A portion of the CCR5 gene spanning the delta32 deletion was amplified by PCR. The resulting DNA fragments were analysed by agarose gel electrophoresis. RESULTS: The rate of HIV-1 infection was correlated strongly with increasing amounts of inoculated clotting factor concentrate. None of the HIV-positive patients (n = 129) had the delta32/delta32 genotype, whereas 12 out of 78 HIV-negative haemophiliacs had the homozygous delta32 deletion. CONCLUSIONS: The delta32/delta32 genotype was highly protective against HIV-1 infection, even in patients who had received millions of non-inactivated clotting factor units. As it is likely that in the early 1980s plasma pools were contaminated not only with monocyte-tropic HIV-1 strains, CCR5 appears to be the major mediator of HIV-1 infection. Furthermore, we conclude that there must be other protective mechanisms in multiply exposed non-infected haemophiliacs who have wild-type CCR5.

Antigen-specific cytokine response to hepatitis C virus core epitopes in HIV/hepatitis C virus-coinfected patients.

OBJECTIVE: Epidemiological data indicate that hepatitis C virus (HCV) infection runs a more rapid and severe course of disease in HIV-coinfected patients, probably because of an altered immune response. DESIGN: We investigated whether HCV-specific cytokine responses are affected by HIV coinfection. METHODS: Using triple colour flow cytometry on peripheral blood lymphocytes after stimulation with the four major immunodominant HCV core T cell epitopes, CT1-CT4, we determined intracytoplasmic production of IFN-gamma, IL-2, IL-4, IL-10 and CD30 expression, a putative surrogate marker of type 2 cells. Fifteen patients with asymptomatic HIV/HCV coinfection (group A), 15 patients with chronic HCV infection (group B) and 10 HIV-infected patients without hepatitis C (group C) were included in the study. RESULTS: In group A, HCV antigens induced significantly higher IL-2 and IFN-gamma production than groups B and C (P < 0.05). Groups A and B showed a similar induction of CD30, which was significantly higher than in group C (P < 0.001). Remarkably, in group A HCV antigens induced IL-4 production in addition to IL-10 and IFN-gamma in the CD30 subset, whereas in groups B and C no IL-4 induction was observed in this T cell subset (P < 0.002). CONCLUSION: Our data suggest that asymptomatic HIV coinfection importantly alters the HCV-specific cytokine response towards a greater production of proinflammatory type 1 cytokines. Moreover, the antiviral activity of type 1 cytokines may be modified by an increased production of type 2 cytokines in the CD30 subset. The altered cytokine pattern may contribute to the adverse natural course of hepatitis C in HIV coinfection.

Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells.

Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfected to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adherent bone marrow stromal cell cultures, established and expanded from iliac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3.84 micrograms/10(6) cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases, the stromal cells were cryopreserved following transfection, and subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 micrograms/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following reinfusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Vector-modified stromal cells were detectable, by polymerase chain reaction (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 x 10(8)), with a total hFIX expression capacity of 281 micrograms/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.

EEG discharges in WR 1-5 HIV-seropositive hemophiliacs.

Human immunodeficiency virus (HIV)-seropositive patients show involvement of the central and/or peripheral nervous system. We present here the results of electroencephalographic (EEG) findings in stage WR 1-5 HIV-seropositive hemophiliacs from a total of 184 who attended our clinic prior to October 1987.

HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII.

Molecular genetic studies have shown that development of antibodies to factor VIII (inhibitors) occurs most frequently in patients with severe haemophilia due to major gene lesions including inversions, stop codons and large deletions. Previous studies of HLA type were performed on inhibitor and non-inhibitor patients with diverse uncharacterized mutations which may have confounded detection of significant associations. We therefore selected a group of patients with a single mutation type, the prevalent intron 22 inversion, with or without inhibitors, to determine HLA genotype. Seventy-one such patients, 42 without and 29 with inhibitors (13 high, 9 low and 7 transient responders) were genotyped for MHC Class I HLA-A, -B, -C and Class II HLA-DQA, -DQB and -DRB loci. No strong correlation of any HLA-allele to inhibitor or non-inhibitor status was found. However, alleles of the haplotype HLA-A3, HLA-B7, HLA-C7, HLA-DQA0102, HLA-DQB0602, HLA-DR15 occurred more often in inhibitor patients. Since the alleles of this extended haplotype are common in the North European population only a very strong association would achieve statistical significance. Further studies of groups of patients similar to those studied here will be needed to confirm or exclude this association.

Sucrose formulated recombinant human antihemophilic factor VIII is safe and efficacious for treatment of hemophilia A in home therapy--International Kogenate-FS Study Group.

To add an increased level of safety to antihemophilic factor replacement therapy, a full-length, recombinant Factor VIII (rFVIII) product has been developed without human-derived plasma proteins during purification and formulation and using an additional solvent/detergent viral inactivation step. This first clinical trial of a sucrose-formulated full-length rFVIII (rFVIII-FS) was conducted in previously treated patients (> or = 100 prior exposure days) with severe (<2% FVIII) hemophilia A in North America (NA) and Europe (EU). Pharmacokinetic profiles for rFVIII-FS were compared with those of currently licensed rFVIII product (Kogenate) in 35 patients. Safety and efficacy during home therapy were evaluated in 71 patients. The new formulation displayed a pharmacokinetic profile similar to that of rFVIII. Patients on home therapy received a cumulative total of 11,867 exposure days, 12,546 infusions, and 22,443,694 IU of rFVIII-FS. Of 2585 bleeds, 93.5% were treated with 1-2 infusions and 80.5% of responses were rated as excellent or good. No evidence of de novo inhibitor formation was observed. Only 0.27% of infusions were associated with any drug-related adverse event. Except for an episode of intermittent chest pain with palpitations which ceased after treatment with analgesics, associated adverse events were mild or moderate. Overall, rFVIII-FS provided excellent hemostatic control, was well-tolerated, and caused no significant adverse effects, thus demonstrating safety and efficacy for treatment of bleeds in patients with hemophilia A.

Pharmacokinetics and tolerability of factor XIII concentrates prepared from human placenta or plasma: a crossover randomised study.

The pharmacokinetics and tolerability of factor XIII (FXIII) from plasma were compared with those of FXIII from placenta in a randomised, double-blind, crossover study involving 13 patients with congenital FXIII deficiency. Both FXIII activity and FXIII antigen were monitored. No difference was seen in the mean half-lives of the two preparations (9.3 days and 9.1 days for plasma and placenta FXIII activity, respectively). Response was similar for both preparations, but was slightly greater for FXIII from plasma (1.6 ormula: see text] vs 1.5 [formula: see text]). Similar results were found for recovery (65% vs 60%). The area under the data completed by extrapolation was significantly higher for FXIII from plasma. No differences between preparations in terms of efficacy or tolerability were observed. It can be concluded that treatment with FXIII concentrate from plasma is as efficient as with FXIII concentrate from placenta in terms of recovery and half-life. Both preparations were equivalent in terms of safety during the observation period. With the administration of monthly injections of approximately 30 U/kg serious bleeding events were prevented and no other serious adverse events occurred.

Haemophilia A: mutation type determines risk of inhibitor formation.

The formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations. large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.

Serological and virological markers of human parvovirus B19 infection in sera of hemophiliacs.

It is known that parvovirus B19 (B19) is transmitted to hemophiliacs by clotting factors prepared from human plasma. However, it is not clear whether B19 is also transmitted by the more recently used inactivated clotting factor preparations. Therefore, we investigated 69 hemophiliacs, mostly children, receiving only virus-inactivated clotting factors. 49 of them (71%) were B19 IgG-positive and 18 of the IgG-positive hemophiliacs (37%) were also B19 IgM-positive. In contrast, out of 73 age-matched controls only 10 (14%) were IgG-positive, two of them being also IgM-positive. In hemophiliacs treated before 1984 with non-inactivated clotting factors, seroprevalence was very similar: 94/136 (69%) presented B19 IgG antibodies as compared to their age-matched controls with 16/50 (32%). Out of the 94 IgG-positive patients 24 (26%) were IgM-positive, whereas IgM antibodies were never found in 16 sera of 16 IgG-positive controls. In 4 out of 24 IgM positive hemophiliacs, B19 DNA was detected in the sera by using the polymerase chain reaction. However, B19 DNA was also found in 3/69 anti-B19 IgM-negative, HIV-infected hemophiliacs (all three patients in CDC stage IV). Since it seems unlikely that the results only represent passive acquisition of B19 DNA from blood products and induction of antibodies by immunization with inactivated antigen, the observations rather suggest that infection with B19 is transmitted by clotting factors, including those treated for virus inactivation.

Inhibitors in German hemophilia A patients treated with a double virus inactivated factor VIII concentrate bind to the C2 domain of FVIII light chain.

To reduce the risk of transmission of hepatitis A virus, an Octapharma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

Congenital deficiency of vitamin K dependent coagulation factors in two families presents as a genetic defect of the vitamin K-epoxide-reductase-complex.

Hereditary combined deficiency of the vitamin K dependent coagulation factors is a rare bleeding disorder. To date, only eleven families have been reported in the literature. The phenotype varies considerably with respect to bleeding tendency, response to vitamin K substitution and the presence of skeletal abnormalities, suggesting genetic heterogeneity. In only two of the reported families the cause of the disease has been elucidated as either a defect in the gamma-carboxylase enzyme (1) or in a protein of the vitamin K 2,3-epoxide reductase (VKOR) complex (2). Here we present a detailed phenotypic description of two new families with an autosomal recessive deficiency of all vitamin K dependent coagulation factors. In both families offspring had experienced severe or even fatal perinatal intracerebral haemorrhage. The affected children exhibit a mild deficiency of the vitamin K dependent coagulation factors that could be completely corrected by oral substitution of vitamin K. Sequencing and haplotype analysis excluded a defect within the gamma-carboxylase gene. The finding of highly increased amounts of vitamin K epoxide in all affected members of both families indicated a defect in a protein of the VKOR-multienzyme-complex. Further genetic analysis of such families will provide the basis for a more detailed understanding of the structure-function relation of the enzymes involved in vitamin K metabolism.

Epidemiologically closely related viruses from hemophilia B patients display high homology in two hypervariable regions of the HIV-1 env gene.

The diversity of human immunodeficiency virus type 1 (HIV-1) is mainly caused by mutations that affect the gene encoding the gp120 envelope protein. Isolates differ to a large extent in the hypervariable regions of gp120. This study was undertaken to determine the degree of variation of HIV-1 env genes isolated from seven individuals with hemophilia B who became infected in association with administration of a suspected clotting factor lot. Two hypervariable regions and part of a constant region from proviral DNA of the peripheral blood leukocytes of these patients were amplified and the products of the polymerase chain reactions were sequenced. The sequences derived from five of the individuals displayed 100% sequence homology, 1 had two and 1 had six deviations from the consensus sequence. The alignment of the amino acid sequence so deduced revealed no comparable homology to any of these two hypervariable regions from a number of published isolates. The genetic variability of HIV-1 seems to be limited, at least in the early phase of infection, allowing the determination of close relationships between epidemiologically related strains.

Parallel evolution in the V3 region of HIV type 1 after infection of hemophiliacs from a homogeneous source.

To determine the genetic diversification in the highly functional V3 loop, we followed up five hemophiliacs who were infected by a homogeneous HIV-1 population from a contaminated clotting factor lot. Initially, all patients displayed identical DNA sequences in this part of the proviral env gene. Therefore, this unique outbreak allows us to investigate the biological and genetic development of a common ancestor virus in different patients. A high degree of homology is maintained in the predominant sequences from 5 until 35 months after seroconversion. Only one patient showed a remarkable diversification 3 years postinfection. However, these genetic changes in the V3 region were not associated with disease progression. Discontinuous sequence changes were observed mainly in a region downstream of the V3 loop. Two positions in particular are involved in a sequence evolution within the V3 loop leading to the same amino acids in different patients. These directed changes occurred at sites that are reported to be critical for the specificity of antibodies (position 308) and viral cytopathicity (position 324). However, the parallel evolution was associated neither with differentiation of the viral phenotype nor with progression of the disease.