LYMPHADENOPATHY-ASSOCIATED VIRUS TYPE 2 IN AIDS AND AIDS-RELATED COMPLEX

128

LYMPHADENOPATHY-ASSOCIATED VIRUS TYPE 2 IN AIDS AND AIDS-RELATED COMPLEX

Clinical and

Virological Features in Four Patients

F. BRUN-VEZINET C. KATLAMA D. ROULOT

L. LENOBLE M. ALIZON

J. J. MADJAR

M. A. REY P. M. GIRARD P. YENI F. CLAVEL S. GADELLE M. HARZIC

Laboratoire de Virologie, Hôpital Claude Bernard; Clinique des Maladies Infectieuses, Hôpital Claude Bernard; Département d’Hépatologie, Hôpital Beaujon; Clinique des Maladies Infectieuses, Hôpital Bichat; Service de Médecine Interne, Hôpital Louis Mourier; Unité d’Oncologie Virale, Institut Pasteur, Paris; Diagnostics Pasteur; Laboratoire de Biologie Cellulaire et Moléculaire, Université A. Carrel, Lyon, France

Lymphadenopathy-associated virus type 2 (HIV 2) was isolated from 3 patients with AIDS and 1 with AIDS-related complex. Clinical features were similar to those in patients infected with HIV 1. Viral isolates were characterised by hybridisation with HIV 1 and HIV 2 DNA probes. HIV 1 and HIV 2 serological studies were performed by enzyme-linked immunosorbent assay (ELISA), western blot, and radioimmunoprecipitation assay. HIV 2 IgG antibodies were detected in all sera. The molecular weights of the most representative HIV 2 proteins were determined by immunoblot. Cross-reactivity was restricted to HIV 1 and HIV 2 core proteins. In all 4 patients the neurotropism of HIV 2 was demonstrated by virus isolation from the cerebrospinal fluid and/or by evidence of intrathecal HIV 2 IgG synthesis. All sera were antibody negative by HIV 1 ELISA. An assay specific for HIV 2 is needed for screening of blood donations and for diagnosis and seroepidemiological study of HIV 2 infection. Summary

Introduction LATELY a new type of human immunodeficiency virus, named lymphadenopathy-associated virus type 2 (LAV 2) or HIV 2, has been isolated from two West African patients with the acquired immunodeficiency syndrome (AIDS).1 HIV 2 showed the same morphology, lymphotropism, and cytopathic effect as HIV 1, but purified viral RNA from these isolates, under stringent conditions, did not hybridise with any DNA probe of HIV 1. Under less stringent conditions, HIV 2 RNA hybridised weakly with the gag and W. M. WATKINS AND OTHERS

REFERENCES—continued

14 Weidekamm E, Plozza-Nottebrock H, Forgo I, Dubach UC. Plasma concentrations of pyrimethamine and sulfadoxine and evaluation of pharmacokinetic data by computerised curve fitting Bull WHO 1982; 60: 115-22 15 Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semi-automated microdilution technique. Antimicrob Ag Chemother 1979; 16: 710-18 16. Charles LJ. Field tnals with chlorproguanil in the prophylaxis of malaria in Ghana.

Bull WHO 1961; 24: 457-63 RE, Gilles HM, Bent NS. A companson of the pyrimethamine and cycloguanil sensitivities of the pre-erythrocytic and erythrocytic stages of drug sensitive and resistant strains of Plasmodium yoelii Ann Trop Med Parasitol 1985;

17. Howells

79: 247-51. 18. Watkins WM, Chulay JD, Sixsmith DG, Spencer HC, Howells RE. A preliminary pharmacokinetic study of the antimalarial drugs proguanil and chlorproguanil. J Pharm Pharmacol (in press). 19 Smalley ME, Brown J. In vitro demonstration of pyrimethamine resistance of’wild’ Plasmodium falciparum in the Gambia Trans R Soc Trop Med Hyg 1982; 76: 324-28 20 Leeds AR, Marston SD. Maternal and foetal malana and E-B virus antibodies following ante-natal chlorproguanil therapy West Afr Med J 1975; 23: 116-19. 21. Neftel KA, Woodtly W, Schmid M, Frick PG, Fehr J. Amodiaquine induced agranulocytosis and liver damage Br Med J 1986; 292: 721-23.

pol regions of HIV 1-the most likely conserved regionsof the genome. Serological investigations showed cross-, reactivity between the envelope antigens of HIV 2 and STLV-III (mac).3 STLV-III (mac) (macaque) is a simian retrovirus very similar or identical to STLV-III (agm) (African green monkey). The HIV 2 envelope glycoprotein its precipitated by the sera of HIV 2 AIDS patients’ sera as well as by the sera of STLV-III infected macaques. Moreover, HIV 2 and STLV-III (mac) envelope glycoproteins have the same molecular weight of about 130-140 kD. So HIV 2 seems more closely related to STLV-III (mac) than to HIV 1. Another HIV, named human T-lymphotropic virus type IV (HTLV-IV), has been isolated from 3 healthySenegalese. Like HIV 2, HTLV-IV shares more common epitopes with STLV-111 (agm) than with the prototype HIV 1 . So far as we know, HTLV-IV is not associated with AIDS or pre-AIDS. We report here HIV 2 isolations and serological studies in AIDS and AIDS-related-complex (ARC) patients. Case-reports Patient 1.- This case has been briefly reported previously.2 The patient was a 32-year-old man born and living on the Cape Verde Islands. Except for being a chronic hepatitis-B-virus carrier he had no medical history until January, 1982, when he presented with severe diarrhoea, fever, and progressive weight loss. He was referred to the Claude Bernard Hospital, Paris, with massive undernutrition (20 kg weight loss). His chronic diarrhoea with numerous cryptosporidia and Isospora belli in stools, together with candidal oesophagitis (June, 1983), met the Centers for Disease Control criteria for AIDS. He denied homosexual activity or intravenous drug abuse and had never had a blood transfusion. In September, 1983, an upper lung infiltrate was detected and Mycobacterium tuberculosis was found in sputum. By October, 1984, despite mild diarrhoea with persistent cryptosporidiosis, his clinical status was much improved and he had gained 10 kg. In December, 1984, cerebral toxoplasmosis was diagnosed with multiple abscesses and this reponded to pyrimethamine and sulfadoxine. From free from diarrhoea and neither found in the stools. In June, his clinical On immunological testing he 1986, showed negative skin tests and low absolute numbers of T4 lymphocytes-from 1984 to December, 1985, T4 < 100/ul and T4/T8 <0-1-but in March, 1986, T4 cells had risen to 1000/ and T4/T8 ratio to 0-9. In August, 1986, he returned to the Cape Verde Islands, where he died a month later. Patient 2.-A single heterosexual man aged 32, originating from Guinea-Bissau, was admitted to the Claude Bernard Hospital in October, 1985, with a twelve-month history of pruritus with prurigo. He had lived in Paris for 5 years but had spent a week in Senegal in 1984. No risk factor for AIDS was found. He had generalised prurigo and paronychia. The relevant biological abnormalities were lymphopenia (1100/pl) and polyclonal hypergammaglobulinaemia (IgG 3-2 g/dl). He was a chronic carrier of HBsAg and was anergic to tuberculin skin tests. HIV serology was negative by enzyme-linked immunosorbent assay (ELISA). His clinical status did not change until February, 1986, when he was readmitted with meningitis and confusion. The cerebrospinal fluid (CSF) showed pleocytosis-76 white blood cells/81 (84% lymphocytes, 9 % neutrophils, 7 % monocytes)—and excess protein (01g/dl). After ten days of ampicillin therapy his clinical status worsened and he had seizures. Computed tomographic (CT) scanning showed several small parieto-occipital hypodense lesions which were enhanced by injection of contrast medium. A further CSF specimen showed aggravation of abnormalities-white cells 260/pl (80% lymphocytes), protein 0-2 g/dl. No improvement was observed after two weeks of antituberculosis therapy. A CT scan revealed enlargement of the lesions indicating brain toxoplasmosis, so instead he was given sulfadoxine and pyrimethamine. Toxoplasma antibodies were present in the serum (IgM 50 IU)

November, 1984, he

cryptosporidium

was

isospora was status was good.

nor

’

129 and the CSF (IgG 10 IU). A skin multitest (Merieux) was negative for all antigens. Total lymphocyte count was 1200/pl with low T4 count (550/µ1) and a low T4/T8 cell ratio of 0-83. His fever abated and the neurological signs disappeared. In June, 1986, his clinical status was good except for weight loss and recurrent oral candidiasis. No other opportunistic infection or evidence of Kaposi’s sarcoma was observed. The CT scan showed only a slightly hypodense occipital zone. In October, 1986, his clinical status was stable. Patient 3.-This patient, aged 34, was born in Senegal near the Guinean border. He has been living in France since 1980. He often travels to Senegal and Guinea-Bissau where his parents live. He was well until 1984 when he presented with watery diarrhoea and a 20 kg weight loss. In February, 1986, he was admitted to a Paris hospital because of chills, fever, and myalgias. He had generalised lymphadenopathy and Salmonella typhimurium was cultured from blood; stool examinations were negative. Despite 14 days’ treatment with intravenous ampicillin and netromicin, S typhimurium septicaemia recurred, but after 21 days of intravenous cefoperazone he became afebrile and improved. He was referred to Beaujon Hospital in April, 1986, with cholestasis and liver cytolysis. Serum HBsAg was then present in serum but IgM HBc and viral DNA were absent. Endoscopic retrograde cholangiography and microscopic examination of choledochal mucosa and liver tissue showed cholangitis. No infectious agent was found. AIDS-related complex was diagnosed on the basis of chronic diarrhoea, > 10 % weight loss, persistent diffuse lymphadenopathy, and normal peripheral blood cell count with low T4 count (297/µ1) and T4/T8 ratio 0-19. The skin tests were negative. Patient 4.-This patient, a 58-year-old Portuguese mother of 13 children, has been living in France since 1970. She had no medical history until 1981 when she presented with asthenia, progressive weight loss (12 kg), Raynaud’s syndrome, xerostomia, and xerophthalmia; she had a polyclonal hypergammaglobulinaemia (2-9 g/dl). She was found to have a cavernous haemangioma of the liver which was removed in September, 1981; the operation required transfusion of seven units of red cells. In September, 1982, Sjogren’s syndrome was confirmed at Louis Mourier Hospital by salivary gland biopsy. In June, 1983, she was assumed to have AIDS because of candidal oesophagitis histologically proven. Skin tests were negative and absolute numbers of T4 lymphocytes were low at 160/gl with T4/T8 ratio 0-17. HIV 1 serology was negative by ELISA. From June, 1983, to September, 1985, she had a series of opportunistic infections-recurrent candidal oesophagitis, chronic diarrhoea, and recurrent herpes zoster infections. In 1984 brain toxoplasma was suspected because of seizures with residual hemiplegia and an isolated but typical temporal abscess on CT scan. Toxoplasmosis antibodies in serum did not change. With sulfadoxine-pyrimethamine therapy the clinical and CT scan signs resolved completely. In September, 1985, she presented with diffuse interstitial pneumonitis. Bronchoalveolar lavage (BAL) showed pleocytosis with 71 % lymphocytes (69% T8). Cytomegalovirus was isolated from BAL fluid but no viral inclusions were found. She improved within two months. From September, 1985, to August, 1986, she had no new opportunistic infection but autoimmune abnormalities were becoming more pronounced. Since 1985 CT scans have shown a generalised cortical atrophy though this is not evident clinically. She is now out of hospital leading a sedentary life and her clinical status is relatively good. Her husband and children are well.

Methods Virus Isolation Retrovirus isolation

was

attempted from

peripheral

blood

lymphocytes and CSF and in patient 1 from bronchoalveolar lavage fluid. The procedure for virus isolation from blood lymphocytes and BAL fluid has been described. 5,6 Briefly, 3 x106 to 5 x 106 mononuclear cells, separated by ’Ficoll’ gradient, were stimulated for 3 days with phytohaemagglutinin (PHA), then cocultivated with PHA-stimulated human peripheral mononuclear cells in RPMI medium supplemented with 10% fetal calf serum, polybrene

(2 µg/ml), L-glutamine (20 mmol/1), penicillin (250

U/ml),

streptomycin (250 ug/ml), and anti human-alpha-interferon. These cultures

were

maintained for 6 weeks. 2 ml of CSF was inoculated

onto PHA-stimulated normal blood mononuclear cells and cultured

in the same conditions. In each case, twice a week, when the medium was changed, viral production was assessed in the cell-free supernatants by measurement of Mg2+-dependent reverse transcriptase (RT) activity. Concurrently T-cell proliferation was evaluated by lymphocyte counts. Isolate from patient 1 was propagated on CEM, a human lymphoid tumour cell line enriched with T4 cells. Cocultivation of a short-term culture of T lymphocytes from a human normal donor infected by the isolate was undertaken with T4-enriched CEM cell line. As assessed by RT activity, continuous production of retrovirus was obtained after 21 days and was used as a source of antigens for HIV 2 serology.

Dot Blot Hybridisation Genomic virion RNA of the isolates was hybridised with HIV 1 and HIV 2 DNA probes in conditions of high stringency. Briefly, virions were pelleted for 7 min at 400 000 g, lysed in NTE-SDS buffer ("tris" 10 mmol/1, pH 7-5, EDTA 1 mmol/1 sodium dodecylsulphate [SDS] 0-1%) and spotted onto nitrocellulose. Spots were made with amounts of virus corresponding to 25 000 cpm of RT activity. The filters were hybridised with an HIV 1 probe nick-translated to a specific activity of 108 cpm/µg, in 50%. fbrmamide,5xx standard saline citrate (SSC), 5 x Denhartat42°C. Filters were washed at 65°C in 0-1 x SSC, 0-1% SDS. Dot blots were also hybridised With an HIV 2 probe derived from a 2 kb cloned cDNA complementary to the genomic RNA from the purified isolate from patient 1; 32P-labelled by nick translation. Hybridisation and washings were conducted in the same conditions as described for HIV 1.

Serology 1 were detected by ’Elavia’ (Diagnostics (Diagnostics Pasteur), and radioimmunoprecipitation assay (RIPA) with 35S-cysteine-labelled HIV 1.6 IgG antibodies to HIV 2 were determined by ELISA, western

IgG antibodies to HIV

Pasteur),

’LAV-Blot’

blot, and RIPA. For ELISA, virus was collected from the supernatants of the infected CEM cell line, pelleted, then purified by banding on a sucrose gradient (20-60 %). Purified virus was disrupted by ’Triton X-100’ (0 1 %). A control antigen was prepared in exactly the same manner as the viral antigen but from uninfected CEM supernatants. Microtitre wells were filled with viral and control antigens at the same protein concentration (1 )ig/ml), diluted in a sodium bicarbonate buffer (50 mmol/1, pH 9-6). Plates were incubated overnight at 20°C, washed with water, and filled with 5 % non-fat dry milk in phosphate-buffered saline. After washing, sera and CSF, diluted 1:100, were added to both antigens and incubated for 90 min at 37°C. After washing, 100 ul of peroxidase-conjugated goat antiserum to human IgG was added to each well for 60 min at 37°C. The enzymatic reaction was carried out with o-phenylenediamine substrate. Sera and CSF were considered positive when the differential optical density (&Dgr;OD) between the two antigens was 03. They were borderline when the AOD ranged between 0 25 and 03. A high HIV 2 positive serum was included on each plate at four dilutions; a standard curve was used for calculation of HIV 2 antibody titres and the ratio of HIV 2 antibodies in sera and CSF was estimated. HIV 2 immunoblot assay was conducted with viral proteins solubilised in 0-625 mol/1 "tris"-HCI pH 68, 1% SDS, 1% 2-mercaptoethanol, and 10 % glycerol, and then heated for 3 min at 95°C and separated on a 12% polyacrylamide gel slab according to Laemmli.7 After completion of electrophoresis, the separated proteins were electrotransferred to a nitrocellulose sheet.8 After the blotting procedure,9 the antigen-antibody reaction was conducted at room temperature, then overnight at 4°C, with sera diluted at 1:100 and CSF diluted at 1:30. A peroxidase-conjugated goat antiserum to human IgG was used. All the washings were performed in phosphate buffered serum containing 0 05 %’Tween20’. Inimunostaining was activated with diaminobenzidine tetrahydrochloride. The reaction was stopped with deionised water.

130

The molecular mass of HIV 2 proteins was determined by comparison with the molecular mass of standards used to calibrate the gel slab-phosphorylase-b 94, albumin 67, ovalbumin 43, carbonic anhydrase 30, tryspin inhibitor 20-1, ot-lactalbuniin 14 4 kD. HIV 2-infected CEM cells were exposed to 35S-cysteine (200 µCi/ml) overnight at 37°C. After collection of the supernatants, the labelled virus was pelleted, then lysed in a RIPA buffer ("tris"-HCI, pH 74 0 05 mol/l, NaCl 0- 15 mol/l, EDTA 1 mmol/1, 1% triton X-100, sodium deoxycholate 0-1%, SDS 0-1%, ’Zymofren’ 5 %). 50 µ1 of the diluted virus (100 000 cpm) was incubated with 5 III serum (dilution 1:10) at 4°C overnight. Immune complexes were adsorbed on ’Protein-A-Sepharose CL4B’ at 4°C for 3 h. After washing, immune complexes were eluted in a sample buffer containing SDS 1 % and heated at 100°C for 3 min. They were then subjected to electrophoresis on a 125% SDSpolyacrylamide slab gel. 35S cysteine proteins were detected by

Fig I-Dot-blot hybridisation of viral genomic RNA from HIV 1 (C) and isolates from patients 2 and 3 with HIV 1 and HIV 2 DNA probes.

autoradiography. Results The results of virological in the accompanying table.

investigations

are

summarised

patient 2, CSF was collected after recovery from brain toxoplasmosis and showed albumin 0-04 g/dl and 3 cells/1. Virus isolation was also attempted from the CSF collected during systematic evaluation of patient 3, with ARC, and patient 4 with AIDS. The cultures did not produce detectable virus. In patient 1, cultured T lymphocytes from BAL fluid twice yielded retrovirus; no other infectious agent was

found.

Viral Isolations

Hybridisation

From the blood lymphocytes of the 4 patients retroviruses were isolated with reverse transcriptase (RT) activity in the culture supernatants exceeding background activity ( < 500 cpm/ml) by from 20 x 103 to 220 x 103 cpm/ml. Beginning between day 10 and 30 after the cocultivation, the viral production was transient, becoming undetectable after two weeks. Before the occurrence of RT activity, the cultures showed enlarged and multinucleated giant-cells and then cytolysis. The retrovirus produced by cultured T lymphocytes from the 4 patients could be readily transmitted to the T4 enriched CEM cells allowing large-scale virus production. Twice a month, fresh T4-CEM cells were added to the infected cell line. In patient 1, viruses were isolated on six occasions from blood lymphocytes and each time they showed very similar RT activity. In patient 2, two isolates were obtained from blood lymphocytes. In patients 1 and 2 retrovirus isolates were easily obtained from the CSF. Patient 1 had no neurological symptoms or signs and the lumbar puncture was done as part of the systematic investigation; CSF analysis showed 0-06 g/dl albumin and 6 lymphocytes/µ1. In

Retroviruses isolated from blood lymphocytes of patients 1, 2, and 3 were further characterised by dot blot hybridisations under high-stringency conditions. Under these conditions, an HIV 1 probe did not hybridise with any of the patients’ isolates. An HIV 2 cDNA probe hybridised strongly to spots from patients 1 (from whom the cloned cDNA was derived) 2, and 3 (fig 1). No hybridisation was detectable with one isolate of HIV 1 from an AIDS patient.

VIROLOGICAL STUDIES IN

PBL

peripheral blood lymphocytes; ND *Judged posmve when &Dgr;OD≽03. tExpressed as 1 dilution. =

=

not

done; S =

serum.

Serology Serological studies of patients 1 and 4 were done with specimens collected from 1983 to 1986 and kept frozen at - 20°C. Sera from patients 2 and 3 were collected in 1986. HIV 1 serology.-No antibody to HIV 1 was detected in sera from the 4 patients. ELI SA gave negative results except in patient 4, whose sera collected in 1986 showed a borderline AOD (0-25). Analysis of these sera by HIV 1 immunoblot assay revealed only weak and inconstant reactivity against some HIV 1 core proteins-p25 and p18-often associated with antibody to the protein p34 encoded by the pol gene. According to the criteria of

4 PATIENTS INFECTED

BY HIV

2

131

CSF

as

against

in sera, the most constant reactivity was observed the proteins of 130-105 kD and 41 kD. Discussion

.

Fig 2-HIV 2 immunoblot analysis, sera and CSF. HIV 1 (-) = control negative sera; HIV 1 (+) = HIV, positive sera from AIDS patient. Strips 1 correspond to samples collected in 1983 and 1986 from patient 1. (S’ = serum 1983; S" serum 1986; C CSF 1986). Strips 2 correspond to samples collected in 1986 from patient 2 (S serum; C CSF). Strips 3 correspond to samples collected in 1986 from patient 3 (S serum. C=CSF). Strips 4 correspond to samples collected in 1983 and 1986 from patient 4 (S’= serum 1983; S"=serum 1986; C=CSF 1986). Strips 5 correspond to sera collected in 1985 and 1986 from the husband of patient 4 (S’= serum 1985; S" = serum 1986). Numbers in margin are the calculated molecular weight (kD) of the most representative HIV 2 viral proteins. an

=

=

=

=

=

positivity for western blots, all these sera would be judged negative. Analysis by radio-immunoprecipitation assay gave the same results. HIV 2 serology.-Tested by HIV 2 ELISA these sera were clearly reactive. The same HIV 2 IgG antibody titres were found in sera collected from 1983 to 1986 in patients 1 and 4. Interestingly, IgG antibodies to HIV 2 were detected in the CSF of all 4 patients. Titres in CSF and sera were calculated on the same running assay. The serum:CSF ratio of HIV 2 IgG antibodies in patients 2, 3, and 4 was < 4, consistent with intrathecal production of HIV 2 IgG antibodies. The possibility of passive transfer from serum was ruled out by the absence of herpes simplex virus antibodies in CSF when high-titre antibodies were present in serum (data not shown). In patient 1 the serum:CSF ratio of HIV 2 IgG antibodies was 40; nevertheless HIV 2 had been isolated from the CSF of this patient. Fig 2 shows immunoblot analysis of sera and CSF from the 4 patients. Serum from a control individual who lacked antibodies to both HIV 1 and HIV 2 did not recognise any specific proteins of HIV 2. A reference HIV 1 positive serum from an AIDS patient recognised HIV 2 proteins of 26 kD. No reactivity was observed with this HIV 1 positive serum against higher molecular mass proteins of HIV 2. All sera of the 4 patients reacted strongly with HIV 2 antigens of 130 kD and 105 kD. These proteins probably correspond to the glycoproteins of HIV 2.2 In all sera (weakly in patient 2) antibody to a 41 kD protein was present. The band appears clearly as a smear that resembles gp41 of HIV 1 (HIV 1 transmembrane glycoprotein). Less prominent reactivity was observed with other proteins of HIV 2-those of approximately 26, 16, 55-53, 68, and 33 kD; proteins of 26 kD and 16 kD are likely to be HIV 2 core proteins; 55-53 consistently appeared as a doublet of bands of equal intensity, with a very similar pattern to HIV 1 p55, precursor of the core proteins. The HIV 2 p68 is analogous to HIV 1 p68, a pol gene product. In patients 1 and 4, sera collected in 1983 and 1986 were compared. In patient 4, the banding patterns are very similar in the two years. In patient 1, strong reactivity against p 16 is seen in the 1986 sample. In

In the 3 AIDS patients with HIV 2 infection the clinical picture was similar to that in AID S patients infected by HIV 1. Opportunistic infections observed were predominantly brain toxoplasmosis, candidal oesophagitis, and chronic diarrhoea with cryptosporidium. None of these patients had Kaposi’s sarcoma. All 3 had profound suppression of cellular immunity. The 4th patient had typical ARC. It is noteworthy that patients 1 and 4, in whom AIDS was diagnosed in 1983, were in reasonable health three years later. This survival time of 1095 days is considerably greater than the median survival time of 261 days from the date of diagnosis for 3204 US AIDS patients.10 A difference in pathogenicity between HIV 1 and HIV 2 seems possible. 3 of the 4 patients originated from West Africa. All denied homosexuality, bisexuality, intravenous drug abuse, or receipt of blood transfusions and none had visited other African countries. In these 3 cases we think it likely that HIV 2 infection was acquired through heterosexual contactswhich is the most probable and usual mode of transmission, of HIV 1 in Africa." In patient 4, a Portuguese woman who has lived in France since 1970, the mode of HIV 2 transmission is somewhat unclear. In this patient, AIDS occurred 2 years after surgery requiring blood transfusions. In 1984 Dr F. Courtois and Dr F. Meyer (Louis Mourier Hospital) traced all the blood donors and all have proved to be seronegative for HIV 2 antibodies. Sera collected in 1985 and 1986 from the patient’s husband were positive for HIV 2 antibody by ELISA and western blot (fig 2). Moreover, in 1986, HIV 2 was isolated from his blood lymphocytes; he has no signs or symptoms of HIV infection. He had never been transfused; he denies sexual contact with any other women; and he has never been to Africa. All 4 children tested (the youngest is 20 years old) were HIV 2

seronegative. HIV 1 and HIV 2 have characteristics in common2-their morphology, their lymphotropism, and their cytopathic effect in vitro. Moreover, the 4 cases indicate that HIV 2 may have the same neurotropism as HIV 1.12,13 In patients 3 and 4 the very low ratio of serum to CSF HIV 2 IgG indicates probable intrathecal production of the antibodies; and patient 4 has cortical atrophy. Immunoblot analysis confirms that HIV 1 and HIV 2 share epitopes of the core proteins-HIV 1, p25, p18, and their precursors p55 and p40; HIV 2, p26, pl6, and possibly p55. We did not find any cross-reactivity between the envelope glycoproteins of the two viruses-gp110, gp41 for HIV 1; gpl30-105 for HIV 2. More accurate chemical studies are needed to defme the precise molecular weight of HIV 2 proteins, particularly the transmembrane glycoprotein. Although HIV 1 and HIV 2 core proteins cross-reacted, none of the HIV 2 seropositive sera we studied (including the serum from the symptomless husband of patient 4) were clearly reactive by HIV 1 ELISA. This could be explained by the decrease of antibodies to core proteins in AIDS patients as shown in HIV 1 infection.14 In fact, 5 out of 20 other HIV 2 seropositive sera collected from symptomless individuals were completely negative by HIV 1 ELISA (F. BrunVezinet, unpublished). In other words, the cross-reactivity between HIV 1 and HIV 2 core proteins does not mean that a screening ELISA test for HIV 1 will detect the presence of antibody to HIV 2.

132 Another lymphotropic virus, HTLV-IV, has been isolated from West African people,; and cross-reactivity between the envelope glycoproteins and the core proteins of HTLV-IV and HIV 2 has been demonstrated.2 But HTLV-IV has no cytopathic effect in vitro. Comparison of the genomic sequences of the viruses will give a precise indication of the relationship. These data, like seroepidemiological studies in progress (C. Katlama, unpublished), indicate that HIV 2 infection is present in France and West Africa. Furthermore, finding that patients infected by HIV 2 can be seronegative by HIV 1 ELISA means that HIV 2 infection should be sought in patients who are HIV 1 seronegative despite clinical evidence of retroviral infection. For the same reason, a specific HIV 2 assay deserves urgent consideration for screening of blood donations. Finally, in West Africa, as elsewhere, clinical virological and epidemiological investigations are needed to determine the prevalence and features of HIV 2 infections. We are grateful to Prof F. Vachon, Prof A. G. Saimot, Prof J. P. Coulaud, Dr J. J. Pocidalo, Prof J. P. Benhamou, Prof D. Picard, and Prof C. Carbon for encouragement and stimulating discussion; Mrs M. Demol and Mrs G. Quairy for technical assistance; and Mrs G. Marjollet for script preparation. This work was supported by grants from I’Universite Paris VII I’Association de la Recherche contre le Cancer, and 1’INSERM U13.

Correspondence should be addressed to F. B.-V., Laboratoire de Virologie, Hopital Claude Bernard, 10 Avenue de la Porte d’Aubervilliers, 75019 Paris, France.

Preliminary Communication

REFERENCES 1. Clavel

F, Brun-Vezinet F, Guetard D, et al. LAV-II. un second retrovirus associé au SIDA en Afrique de l’ouest. CR Acad Sci Pans 1986; 13: 485-88 2. Clavel F, Guetarad D, Brun-Vezinet F, et al. Isolation of a new human retrovirus from west African patients with AIDS. Science 1986; 233: 343-46. 3. Daniel MD, Letvin NL, King NW, et al Isolation of T-cell tropic HTLV-III-like retrovirus from macaques. Science 1985; 228: 1201-04. 4. Kanki PJ, Barin F, M’Boup S, et al. New human T-lymphotropic retrovirus related to simian T-lymphotropic virus type III (STLV-III). Science 1986; 232: 238-43 5. Barré-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immunodeficiency syndrome (AIDS). Sceince 1983; 220: 868-70. 6. Ziza JM, Brun-Vezinet F, Venet A, et al Lymphadenopathy-associated virus isolated from bronchoalveolar lavage fluid in AIDS-related complex with lymphoid interstitial pneumonitis. N Engl J Med 1985; 313: 183. 7. Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-85. 8. Khyse-Andersen J Electroblotting of multiple gels. a simple apparatus without buffer-tank for rapid transfert of proteins. J Biochem Biophys Meth 1984, 10: 203-09. 9. Johnson DA, Gautsch JW, Sportman JR, Elder JH. Improve technic utilizing non-fat dry milk for analysis of proteins and nucleic acids transfert to nitrocellulose Gene Ann Techn 1984; 1: 3-8. 10. Rothenberg RB, Woelgel MW, Fech G, Krystal A, Milberg J, Stonebruner R Survival with acquired immunodeficiency syndrome. International Conference on AIDS, Paris, June, 1986 11 Piot P, Quinn TC, Taelman H, et al Acquired immunodeficiency syndrome in a heterosexual population. Lancet 1984; ii: 65-69. 12. Levy JA, Shimabukuro J, Hollander J, Mills J, Kaminsky L. Isolation of AIDS associated retrovirus from cerebrospinal fluid and brain from patients with neurological symptoms. Lancet 1985; ii: 586-88. 13. Goudsmit J, Wolters EC, Bakker M, et al. Intrathecal synthesis of antibodies to HTLV-III in patients without AIDS or AIDS related complex Br Med J 1986, 292: 1231-34. 14. Groopman JE, Chen FW, Hope JA, et al. Serological characterization of HTLV-III infection in AIDS and related disorders. J Infect Dis 1986; 153: 736-42

survival of 4.2 months.1 3’-azido-3’-deoxythymidine (AZT) is a potent inhibitor of HIV replication in vitro,2and if it crosses the blood-brain barrier we might expect such patients to respond to antiviral therapy. In our open studies of AZT in AIDS and AIDS-related complex, a patient with peripheral neuropathy attributed to HIV lost his symptoms after treatment.3 We report here four additional patients with HIV-associated neurological abnormalities treated with AZT.

mean

RESPONSE OF HUMANIMMUNODEFICIENCY-VIRUS-ASSOCIATED NEUROLOGICAL DISEASE TO 3’-AZIDO-3’DEOXYTHYMIDINE ROBERT YARCHOAN GARY BERG PIM BROUWERS MARGARET A. FISCHL A. ROBERT SPITZER ALISON WICHMAN GRAFMAN ROSE V. THOMAS JORDAN SAFAI ARTURO BRUNETTI BIJAN CARLO F. PERNO PETER J. SCHMIDT STEVEN M. LARSON CHARLES E. MYERS SAMUEL BRODER National Cancer Institute, Department of Nuclear Medicine of the National Institutes of Health Clinical Center, National Institute of Mental Health, and National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland, USA; University of Miami, Florida; and Memorial Sloan-Kettering Cancer Center, New York

patients with human-immunodeficiency-virus-associated neurological disease were treated with 3’-azido-3’-deoxythymidine (AZT). Three (two with chronic dementia, and one with chronic dementia and peripheral neuropathy) improved as assessed by clinical examination, psychometric tests, nerve conduction studies, and/or positron emission tomography; there was no improvement in the fourth patient who presented with paraplegia. These results support the hypothesis that certain AIDS-virus-associated neurological abnormalities are reversible by antiretroviral chemotherapy.

Summary

Four

INTRODUCTION

NEUROLOGICAL manifestations of AIDS signal a poor prognosis; patients with AIDS-related dementia have a

PATIENTS AND METHODS

The patients gave informed consent to the investigation and the protocol was approved by the National Cancer Institute

institutional review board. Each patient was assessed medically, neurologically, and immunologically before and during administration of AZT. For neuropsychological assessment we used trailmaking tests and the WAIS-R digit-substitution test as measures of attention; the Wechsler memory stories test and Kimura recurring figures test as measures of memory; and the Purdue or grooved pegboard test as a measure of coordination.’ Nerve conduction tests were done with standard methods, with the limbs at a constant temperature. Positron emission tomography (PET) was done with 2[18]fluoro-2-deoxy-d-glucose (FDG) and a standardised protocol5 Briefly, subjects were given an intravenous injection of 5 mCi FDG (1mCi = 3-7 x 107 Bq), and arterial blood samples were taken froma radial artery catheter at timed intervals. After 45 min of radionuclide uptake, during which the patient rested on the scanner bed with both eyes covered, PET scans were obtained with the NIH ’Scanditronix PC-1024’ scanner. The images were corrected for attenuation of emitted radiation with the ellipse border method. RESULTS

Two patients with chronic dementia and one with chronic dementia and peripheral neuropathy improved after treatment with AZT; there was no improvement in the fourth patient who had paraplegia.