Low incidence of hepatitis B virus reactivation and subsequent hepatitis in patients with chronic hepatitis C receiving direct-acting antiviral therapy.

To determine the clinical characteristics of hepatitis B virus (HBV) reactivation in patients undergoing interferon-free antihepatitis C virus (HCV) therapy, we examined HBV DNA in 25 HBV co-infected patients and 765 patients with resolved HBV infection during and after treatment with direct-acting antiviral agents (DAAs). Among those with HCV genotype 1, asunaprevir plus daclatasvir was administered to 160 patients, sofosbuvir (SOF) plus ledipasvir to 438 patients and paritaprevir plus ombitasvir and ritonavir to 25 patients. In total, 167 patients with genotype 2 were treated with SOF plus ribavirin. Three patients with an HBV DNA level ≥2000 IU/mL were treated with entecavir before anti-HCV therapy, without reactivation of HBV. In 3 of 22 (12%) HBV surface antigen (HBsAg)-positive patients with an HBV DNA level <2000 IU/mL, the viral load increased during treatment. However, hepatitis flare did not occur in these patients. There was no significant difference in clinical history between patients with and without HBV reactivation. Among 765 patients with resolved HBV infection, HBV reactivation occurred in 1 (0.1%) patient after initial resolution, whose HBV DNA level spontaneously decreased after DAA therapy. We compared anti-HBs titres at baseline with those at post-DAA therapy in 123 patients without HBsAg. There was no significant difference in anti-HBs levels between the two points (P = .79). In conclusion, HBV reactivation was rare in HBsAg-negative patients treated with DAA therapy. Additionally, hepatitis did not occur in HBV-reactivated patients with a baseline HBV DNA level <2000 IU/mL before DAA therapy.

The identification of sandfly species, from an area of Argentina with endemic leishmaniasis, by the PCR-based analysis of the gene coding for 18S ribosomal RNA.

The area around Río Blanco, in the Orán department in the north of the Argentinian province of Salta, is endemic for American tegumentary leishmaniasis. In an attempt to facilitate the identification of the Lutzomyia species in this area, sequences of the gene coding for the 18S ribosomal RNA (rRNA) of sandflies caught in a Shannon trap were explored, by a combination of PCR and analysis of restriction-fragment-length polymorphism (RFLP). The products from the PCR, which employed two primers developed specifically for this study (Lu.18S 1S and Lu.18S AR), were cloned into a commercial vector (pGEM-T Easy) so that their nucleotide sequences could be investigated. In the RFLP analysis, the products of single and double digestion with the AfaI and HapII restriction enzymes were separated by electrophoresis in 3% or 4% agarose. Taken together with the results of a morphological investigation of the flies, the resultant DNA fragment patterns were sufficient to identify most of the sandflies caught as Lu. neivai. Although two other species, Lu. cortelezzii and Lu. sallesi, were collected, they were relatively rare and only identified morphologically. A single digestion of the 18S-rRNA gene sequences with AfaI or HapII appeared sufficient and useful for the identification of Lu. neivai from the north of Salta province, and for several other Lutzomyia species.

Morphological observations and the effects of artificial digestive fluids on the survival of Diploscapter coronata from a Japanese patient.

Unusual non-human parasitic nematodes and eggs were detected in the faeces of an 8-year-old Japanese female suffering from Henoch-Schönlein purpura. The worms were adult female rhabditiform nematodes measuring 325.6-441.2 micro m in length and 18.3-26.5 micro m in width. One pair of the labia oris was notched with many spiny projections, while the other pair was strongly curved outwards. The worms were identified using light and scanning electron microscopy as the free-living nematode Diploscapter coronata (Cobb) based on their characteristic morphology. The patient's faeces containing worms and eggs were cultured using a filter-paper culture technique and after 7 days of culture, male as well as female worms were recovered. Worm survival time and hatchability of the eggs were examined in vitro after treatment with an artificial gastric or intestinal fluid. Although adult worms survived for less than one minute, eggs hatched after treatment with artificial gastric fluid. This suggests that eggs accidentally ingested or produced by adult D. coronata could develop in the human gastro-intestinal tract. Some morphological features of male D. coronata are also described.

Zoonotic onchocerciasis caused by a parasite from wild boar in Oita, Japan. A comprehensive analysis of morphological characteristics of the worms for its diagnosis.

Histological examination of a nodule removed from the back of the hand of a 58-year-old woman from Oita, Kyushu, Japan showed an Onchocerca female sectioned through the posterior region of the worm (ovaries identifiable) and young (thin cuticle). Six Onchocerca species are enzootic in that area: O. gutturosa and O. lienalis in cattle, O. suzukii in serows (Capricornis crispus), O. skrjabini and an Onchocerca sp. in Cervus nippon nippon, and O. dewittei japonica in wild boar (Sus scrofa leucomystax). Diagnostic characters of female Onchocerca species, such as the cuticle and its ridges, change along the body length. Tables of the histologic morphology of the mid- and posterior body-regions of the local species are presented. In addition, it was observed that transverse ridges arose and thickened during the adult stage (examination of fourth stage and juvenile females of O. volvulus). The specimen described in this report, with its prominent and widely spaced ridges, was identified as O. d. japonica. Four of the 10 zoonotic cases of onchocerciasis reported worldwide were from Oita, three of them being caused by O. d. japonica, the prevalence of which in local wild boar was 22 of 24 (92%).

Type I interferon receptor and response to interferon therapy in chronic hepatitis C patients: a prospective study.

The type I interferon (IFN) receptor consists of at least two subunits, IFNAR1 and IFNAR2. We previously found a correlation between IFNAR1 and IFNAR2 expression in liver, and a correlation in IFNAR2 expression, but not in IFNAR1, between liver and peripheral blood mononuclear cells (PBMCs). The aim of this study was to prospectively assess whether IFNAR2 expression levels in PBMCs as well as in liver act as markers for predicting response to IFN therapy in chronic hepatitis C patients. Fifty-two Japanese patients with chronic hepatitis C, were enrolled. IFNAR2 mRNA was quantified using competitive polymerase chain reaction, in liver and PBMC specimens, and of the 52 patients assigned to receive a 6-month course of interferon-alpha therapy, 36 patients who received more than 300 million units of interferon were analysed. IFNAR2 mRNA expression levels were significantly higher in liver than in PBMCs in all 36 patients (P = 0.016). Seventeen sustained virologic responders showed lower pretreatment hepatitis C virus (HCV)-RNA levels (P = 0.017) in serum and higher pretreatment levels of IFNAR2 mRNA in liver (P = 0.007), but not in PBMCs, compared with nonsustained virologic responders. In multivariate analysis, these factors were independently associated with a sustained virologic response (i.e. HCV-RNA level: odds ratio 0.23, 95% CI 0.038-0.864; and IFNAR2 in liver: odds ratio 1.116, 95% CI 1.015-1.227). Hence, IFNAR2 expression levels in liver, but not in PBMCs, is predictive of response to IFN treatment in chronic hepatitis C patients.

Interferon retreatment reduces or delays the incidence of hepatocellular carcinoma in patients with chronic hepatitis C.

Inhibition of hepatocarcinogenesis is a crucial issue in treating chronic hepatitis C patients, especially those who do not respond completely to interferon therapy. Interferon has been reported to reduce the incidence of hepatocellular carcinoma (HCC) not only in sustained virological responders but also in transient biochemical responders. However, the incidence of HCC increases in 5 years or more after interferon therapy in transient biochemical responders. The aim of this study is to assess whether interferon retreatment reduces the incidence of HCC in chronic hepatitis C patients in whom hepatitis C virus was not eradicated during initial interferon therapy. We enrolled 309 patients who were not sustained virological responders after initial interferon treatment consisting of a total dose of more than 250 megaunits of interferon and were followed for more than 2 years after treatment. Ninety-nine patients received interferon retreatment and 210 did not. Two courses of interferon therapy were administered in 84, three courses in 14 and five courses in one. The incidence of HCC was compared between patients with retreatment and those without. In the clinical characteristics, retreated patients were younger and followed up for a longer time period. The cumulative incidence of HCC was significantly lower in retreated patients. In multivariate analysis, patients' age (P=0.018) and the number of courses of interferon therapy (P=0.022) were independently associated with HCC incidence. These results suggest that interferon retreatment reduces or delays the incidence of HCC in chronic hepatitis C patients who did not completely respond to initial therapy.

Constrained genomic and conformational variability of the hypervariable region 1 of hepatitis C virus in chronically infected patients.

We analysed the genomic and conformational variability of the hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) to evaluate the importance of its biological role. A total of 865 genotype 1b HVR1 subclones were collected from serially sampled sera in 11 patients with chronic hepatitis C, four of whom received interferon therapy. Consequently, 169 distinct sequences were examined for amino acid substitutions as well as hydrophilic or hydrophobic profile at each amino acid position within HVR1. Secondary structure of HVR1 was also predicted by the method of Robson in 90 distinct sequences from eight patients, including three interferon-treated patients. Some positions within the HVR1 were invariable or nearly so as to amino acid substitution. Hydrophilic or hydrophobic residues exclusively predominated at several positions. These constrained amino acid replacement and hydrophilic or hydrophobic profiles were conserved irrespective of interferon therapy, though the frequency of amino acid replacement was greater at almost all amino acid positions within the HVR1 in interferon-treated patients. The quasispecies of HCV showed various secondary structures of HVR1, but many sequences seemed to have common characteristics. beta sheet conformations around both the N-terminus and position 20 (numbered from the NH2 terminus of E2 envelope glycoprotein), and/or coil structures around the C-terminus of HVR1 could be identified. These results suggest that HVR1 amino acid replacements are strongly constrained by a well-ordered structure, in spite of being tolerant to amino acid substitutions, and imply an important biological role of the HVR1 protein in HCV replication.

A possible role of hypervariable region 1 quasispecies in escape of hepatitis C virus particles from neutralization.

We examined serial changes in the hypervariable region 1(HVR1) quasispecies both in immune and nonimmune complexed hepatitis C virus (HCV) particles from 12 patients with chronic hepatitis C to elucidate the mechanism by which genetic diversification of HCV during the course of infection allows escape of virus from the humoural immune response. Immune and nonimmune complexes were separated by differential flotation centrifugation and immunoprecipitation, and their HVR1 quasispecies were determined by subcloning and sequencing. The presence of a specific antibody against a specific viral clone in serum was examined in two patients by Western blotting of the corresponding recombinant HVR1 protein. The distribution of HVR1 quasispecies in both immune and nonimmune complexes conspicuously changed over time in most of the patients studied. In seven patients, various HCV clones serially shifted from nonimmune complexes to immune complexes. In four of them, a group of clones with similar HVR1 sequences to each other remained predominant in nonimmune complexes, whereas minor clones with sequences considerably divergent from the predominant clones shifted from nonimmune complexes to immune complexes. These results suggest a mechanism for persistent infection of HCV, in which major HCV clones escape from neutralization by anti-HVR1 antibodies by generating considerably divergent minor 'decoy' clones which may be preferentially neutralized.

[A possible role of human leukocyte antigen(HLA) typing for predicting response to interferon therapy in chronic hepatitis C].

Hepatitis C viral load, genotype and/or staging of liver fibrosis are known to be factors for predicting response to interferon(IFN) therapy in patients with chronic hepatitis C. The aim of this study is to investigate if human leukocyte antigen(HLA) typing is related to the response to IFN therapy. The seventy six Japanese patients were studied and categorized into two groups: 46 patients with chronic hepatitis C (Group A) and 30 with liver disease unrelated to HCV infection(Group B). In addition, 39 patients who were treated with IFN were classified into complete responders(CR) and non-complete responders (NR). There was not any differences in HLA typing between group A and B, but the frequency of HLA class I B51(5) was higher in CR than in NR patients(p = 0.045). When restricted to those who had low viral load(under 10(55) copies/ml) and genotype 2a or 2b, HLA class I CW1 was found in 7 responders(70%) and in 1 non-responder(14%) (p = 0.023). HLA class II DR9 was not found in responders but in 3 non-responders(p = 0.022). These preliminary results suggest that HLA types may be related response to IFN therapy in patients with chronic hepatitis C.

Impaired protective immunity and T helper 2 responses in alymphoplasia (aly) mutant mice infected with Trichinella spiralis.

The alymphoplasia (aly) mutation of mice prevents the development of systemic lymph nodes and Peyer's patches. The mutant homozygotes (aly/aly) are partially deficient in both humoral and cell-mediated immune functions. In the present study, we show that adult worm expulsion was slightly delayed and that T helper 2 (Th2)-type responses were partially defective in aly/aly mice after infection with Trichinella spiralis. Male aly/aly and aly/+ mice (8-weeks old) were infected with 400 muscle larvae. There was no difference in worm recovery between the two groups on day 5. However, worm recovery in aly/aly mice was significantly higher than that in aly/+ mice on day 14. Mucosal mast cells increased in number and peaked 14 days after infection in aly/+ mice. aly/aly mice were deficient in their mucosal mast cell response through out the primary infection. To examine the existence of mast cell precursors, aly/aly mice were treated with recombinant interleukin-3 (rIL-3) before infection. The mast cell response was poorly induced in aly/aly mice treated with rIL-3. An immunoglobulin E (IgE) response was not detected in aly/aly mice during the course of infection. Serum IgG1 levels in aly/aly mice were significantly lower than that of aly/+. The serum IgG2a levels increased in both strains of mice. However, IgG2a production in aly/aly mice on day 14 was half as much as that in aly/+mice. Stimulation of splenic T cells in vitro with anti-CD3 monoclonal antibody (mAb) showed that spleen cells from aly/+ mice on day 5 produced more IL-4 than spleen cells from aly/aly mice. IL-4 production from aly/aly mice on day 14 was half that from aly/+ mice. Interferon-gamma (IFN-gamma) was produced in both aly/aly and aly/+ mice on day 14. Proliferation assay showed that T cells of aly/aly mice responded poorly when cultured with antigen-presenting cells. These results suggest that aly gene is needed for the induction of protective immunity and Th2 responses in mice infected with T. spiralis.

Characterisation of Bangladeshi Leishmania isolated from kala-azar patients by isoenzyme electrophoresis.

To identify the prevalent Leishmania species in Bangladesh, a total of nine patients aged 4-35 years, were studied; six (66.7%) of them were below 20 years of age. All the patients were clinically diagnosed to have visceral leishmaniasis; their haematological profile was in accordance with leishmaniasis and all were improved after treatment with sodium stibogluconate. All the aspirated materials (eight bone marrows and one splenic aspirate) yielded growth of Leishmania parasite in NNN media; Leishman-Donovan bodies were found in seven (77.8%) of them in a Giemsa stained smear. Aldehyde test (AT) was positive in all the nine cases examined, whereas, complement fixation test (CFT) was positive in seven (77.8%) and indirect fluorescent antibody test (IFAT) in eight (88.9%) cases. In this study, five of the nine isolates from kala-azar patients were characterised by isoenzyme analysis comparing with five WHO reference strains viz., Leishmania (Leishmania) donovani (DD8), L.(L.) donovani (HU3), L.(L.) infantum (IPT-1), L.(L.) tropica (K-27) and L.(L.) major (5-ASKH) using cellulose acetate electrophoresis. By analysing 11 soluble isoenzymes it was found that all five WHO reference strains had distinct electrophoretic mobility of the isoenzymes studied. No interspecies difference was observed amongst the five isolates from kala-azar patients examined and their isoenzyme profiles were consistent with WHO reference strain of L.(L.) donovani (DD8) but different from L.(L.) donovani (HU3).

Successful radiofrequency catheter ablation from the supravalvular region of the aortic valve in a patient with outflow tract ventricular tachycardia.

Outflow tract ventricular tachycardia (OT-VT) was successfully ablated from the right coronary cusp of the aortic valve. The 12-lead ECG was totally different from the typical right ventricular OT-VT because the R/S ratio in precordial lead V1 was equal to 1 and tall R waves in precordial leads V2-6 were seen. Radiofrequency energy application from the right coronary cusp of the aortic valve successfully ablated this VT without complications. Radiofrequency catheter ablation from the right coronary cusp of the aortic valve can be done safely and effectively.

GB virus C/hepatitis G viremia and antibody response to the E2 protein of hepatitis G virus in hemodialysis patients.

The aim of this study was to assess the relationship between the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) RNA and that of antibody to the putative E2 protein (anti-E2) in hemodialysis patients. GBV-C/HGV RNA in serum was detected by a reverse transcription polymerase chain reaction (RT-PCR) assay, and anti-E2 was measured in 244 hemodialysis patients by enzyme-linked immunosorbent assay using recombinant E2 protein. The GBV-C/HGV RNA level was determined by competitive RT-PCR with an interval of 1 year. GBV-C/HGV RNA, anti-E2. and both together were detected in 11 (4.5%), in 19 (7.8%), and in 3 patients (1.2%), respectively. Comparison of clinical characteristics between GBV-C/HGV RNA-positive patients and negative patients revealed the longer duration of hemodialysis (9.8 years vs. 6.0 years; p < 0.05), and the greater frequency of anti-hepatitis C virus (HCV) (63.6% vs. 20.3%; p < 0.05) and HCV RNA (36.4% vs. 12.9%; p < 0.05) in GBV-C/HGV RNA-positive patients. The GBV-C/HGV RNA levels of patients who were positive for anti-E2 remained under detection limit (< 10(2) copies/mL), whereas only one of eight patients who were negative for anti-E2 showed a GBV-C/HGV RNA level under detection limit (p < 0.05). The presence of anti-E2 in serum was associated with loss of detectable GBV-C/HGV RNA or with a very small amount of HCV RNA in hemodialysis patients.

Hepatitis C virus quasispecies and response to interferon therapy in patients with chronic hepatitis C: a prospective study.

We prospectively examined whether the complexity of hepatitis C virus (HCV) quasispecies is related to the response to interferon (IFN) therapy. Among 64 patients who had histologically proven chronic hepatitis and were treated with natural IFN-alpha, 53 patients were analysed. The other 11 patients discontinued therapy because of adverse effects of IFN. The complexity of the hypervariable region 1 (HVR 1) in quasispecies was determined using both clone number determined by fluorescence single-strand conformation polymorphism (SSCP) and nucleotide diversity determined by direct sequencing. These parameters were measured not only before treatment but also at completion and 6 months after therapy, if serum HCV RNA was detectable. This population of patients was different from the general Japanese population with regard to the high prevalence of patients infected with genotype 2a or 2b (49%), who had a higher viral load than those with genotype 1b (P = 0.021). Twenty-two patients (41.5%) were sustained responders. Genotype non-1b (P = 0.0009) and a smaller clone number (P = 0.008) were significantly associated with a sustained response. In multivariate analysis, these variables were independently associated with a sustained response (i.e. genotype: odds ratio 6.84, 95% CI 1.84-30. 12; and clone number: odds ratio 1.26, 95% CI 0.99-1.68). The clone number and nucleotide diversity did not change significantly between pretreatment and at completion or 6 months after therapy. These results suggest that lower complexity of HVR 1 quasispecies predicts a preferable response to IFN therapy that is independent of viral load, especially in the population of the relatively high prevalence of patients infected with genotype 2.

Factors predicting progression to cirrhosis and hepatocellular carcinoma in patients with transfusion-associated hepatitis C virus infection.

The clinical progression of chronic hepatitis C is not uniform throughout the entire period of infection and is more rapid in patients with advanced histologic disease. Our study was designed to identify factors contributing to progression to cirrhosis and hepatocellular carcinoma by taking the entire period of infection into consideration. Two hundred thirteen patients with transfusion-associated hepatitis type C chronic liver disease were included in this study. They did not have either a history of antiviral therapy or any other potential causes of chronic liver disease except for transfusion. Hepatitis C virus genotype 1b was detected in 144 (68%) patients, followed by 2a in 51 (24%), 2b in 11 (5%), 1a in 4 (2%), and coinfection with 1b and 2a in 3 (1%). The log-rank test in the Kaplan-Meier method revealed that the cumulative percentage of cirrhosis-free or hepatocellular carcinoma-free patients became significantly lower as the transfusion age went up. Patient age at the time of transfusion was the only independent factor related to disease progression in multivariate analysis using Cox's proportional hazards model. Thus age at transfusion should be taken into consideration in designing the optimal follow-up schedule and therapy in patients with posttransfusion-associated chronic hepatitis C.

Differences in hypervariable region 1 quasispecies of hepatitis C virus in human serum, peripheral blood mononuclear cells, and liver.

Hepatitis C virus (HCV) has been reported to potentially replicate in peripheral blood mononuclear cells (PBMCs), but direct information on the pathogenic implication of HCV infection in PBMCs is still limited. To investigate this issue, we compared the complexity of HCV quasispecies in serum, PBMCs, and livers of 13 patients with type C chronic liver disease. Hypervariable region 1 (HVR 1) was amplified by reverse-transcription polymerase chain reaction (RT-PCR), and the PCR products were subcloned and sequenced. Considerable differences in the complexity of HVR 1 quasispecies were found in the serum, PBMCs, and liver in all patients, and the predominant sequences from each source were mutually different in 3 (23%) patients. An amino acid sequence unique to each source existed as well as a sequence common to serum and PBMCs, common to serum and livers, or common to PBMCs and liver. These results suggest infection of PBMCs by HCV, and that HCV in PBMCs may be differently exposed to host immunity from that in liver.

Use of urine samples from healthy humans, nephritis patients or other animals as an alternative to foetal calf serum in the culture of Leishmania (L.) donovani in vitro.

The effect of supplementing in-vitro cultures of Leishmania donovani with urine was investigated. The parasites were isolated from Bangladeshi patients with visceral leishmaniasis. The urine samples used were collected from healthy human donors, patients with nephrotic syndrome, diabetic nephritis (DN) or diabetes mellitus, a dog and a cow. Promastigotes from blood-agar cultures were inoculated into RPMI-1640 basal medium with 10% heat-inactivated foetal calf serum (FCS) and/or 1%-20% urine. The parasites were then counted in a haemocytometer, on days 2, 4, 5, 6, 7, 8, 10, 12 and 14 post-inoculation. From day 4, the numbers of parasites/ml in cultures containing 5% healthy-human urine but no FCS were at least as high as those in cultures containing 10% FCS but no urine (P = 0.191). The wet weights of parasites harvested from mass cultures of the parasites in RPMI-1640 plus 5% healthy-human urine and in RPMI-1640 plus 10% FCS were practically the same. Multiplication of the parasites in the presence of 5% urine from a DN patient was significantly greater (P < 0.01) than that seen with other urine samples at the same concentration or with 10% FCS. The multiplication seen with 8% canine urine was almost the same as with 5% healthy-human urine. Parasites could be maintained in RPMI-1640 plus 5% healthy-human urine for at least 40 days, sub-culturing every 4 days. Urine may be a better and much cheaper stimulant of Leishmania multiplication in vitro than FCS.

Exogenous interleukin-3 enhances IL-4 production by splenic CD4+ cells during the early stages of a Trichinella spiralis infection.

Treatment with recombinant interleukin-3 (rIL-3) augmented IL-4 production of spleen cells in mice infected with Trichinella spiralis. In a previous report, we showed that treatment with rIL-3 accelerated IgE responsiveness in mice. We have examined IL-4 and interferon (IFN)-gamma production by spleen cells from both rIL-3-treated and untreated mice during the early stages of infection. The results indicated that IL-4 production was enhanced in rIL-3-treated mice compared to that in untreated mice. In contrast, there was no difference in IFN-gamma production between the two groups. Augmentation of IL-4 production was dependent on the dose of rIL-3 injected before infection. To examine if the treatment with rIL-3 affects T cell function, spleen cells from mice treated with various doses of rIL-3 were cultured under the stimulation with anti-CD3 (T cell receptor complex) mAb and then assessed for cytokine production. IL-4 production increased depending on the dose of rIL-3, while IFN-gamma production did not. Furthermore, spleen cells were separated by surface markers, Thy1.2, CD4 and CD8. Thy1. 2+ cell population responded significantly to produce IL-4 after anti-CD3 stimulation, when compared with IL-4 production of Thy1.2- cell population. A major producer of IL-4 in T cells was CD4+ cell population but not CD8+ cell population. IL-4 production was suppressed in rIL-3-treated mice injected with anti-CD4 mAb. These results suggest that IL-3 might play a role as Th2 amplifier in immune response to parasite infection.

Differences in hypervariable region 1 quasispecies between immune complexed and non-immune complexed hepatitis C virus particles.

Antibody to the hypervariable region 1 of hepatitis C virus (HCV) is thought to have neutralizing activity. The complexity of hypervariable region 1 quasispecies was compared between immune complexed and non-immune complexed HCV particles. Immune complexes and non-immune complexes, including intact HCV virions, were separated by differential flotation centrifugation and immunoprecipitation, and immune complexes were observed in 9 of 11 patients with chronic hepatitis C. Considerable differences in both hypervariable region 1 quasispecies and predominant clones were demonstrated between immune complexes and non-immune complexes in 4 patients and not in 5 patients. These results suggest that the specificity of antibody to hypervariable region 1 is related to the formation of immune complexes and that escape mutants with highly different quasispecies are resistant to neutralization by antibody to hypervariable region 1.