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LYTIC INFECTION BY BRITISH AIDS VIRUS AND DEVELOPMENT OF RAPID CELL TEST FOR ANTIVIRAL ANTIBODIES
695
The trial reported here was a pilot study to find out whether a combination of mefloquine with other anti-malarials is as efficient as expected of a long-acting schizonticide to which widespread resistance has not yet developed. Combining mefloquine withSP has been said to delay emergence of resistant strains in P berghei isolates that are chloroquinesensitive or partially resistant to either mefloquine5 or SP. It thus seems sensible for mefloquine to be used only in combination in countries such as Burmawhere a proportion of P falciparum may still be sensitive to SP, so as to prolong the operational life of this drug to a maximum. Another advantage of giving mefloquine in combination is that a smaller dose of the drug will suffice, for therapy or prophylaxis. Cost is extremely important when large populations require a drug, especially an expensive one such as
mefloquine.
SP that was twice that taken before the trial was probably because exposure to malaria was higher during than before the trial. Our findings support the view that MSP should be used for prophylaxis in special high-risk groups continually exposed to multidrug-resistant falciparum malaria. The other drugs now available are so inefficient that their continued use for such groups can no longer be justified. This study was conducted with permission and support from the Directorate of Medical Services, Ministry of Defence. Mefloqume for this study was supplied by Mepha Pharmaceutical Specialties, Basle, Switzerland. Correspondence should be addressed to K. W. REFERENCES
M, Campbell CC, Freeman J, Doberstyn EB, Brandling-Bennett AD. Drug therapy for Plasmodium falciparum malaria resistant to pyrimethamine-sulfadoxine (Fansidar). Lancet 1981, ii: 1066. Hurwitz ES, Johnson D, Campbell CC. Resistance of Plasmodium falciparum malaria
1. Reacher
2.
The group that received MSP became almost completely free from falciparum malaria, whereas in those who received SP the blood film positivity rates for falciparum malaria were high-43’ 66% in group 1 and 70, 37% in group 2. The fact that their positivity rates rose during the trial despite a dose of
to
sulfadoxine-pyrimethamine (Fansidar)
in
a
refugee
camp
in
Thailand. Lancet
1981; i 1068-70. 3. 4 5.
Report of the Fourth Scientific Working Group on the Chemotherapy of Malaria (CHEMAL) WHO Tech Rep Ser no 711, 1984. Peters W. Antimalarial drug resistance. Br Med Bull 1982; 38: 187-92 Report of the Steering Committees ofthe Scientific Working Groups on Malaria. June, 1980-June, 1983. TDR/MAL/SC-SWG (80-83)/83.3.
Methods and Devices LYTIC INFECTION BY BRITISH AIDS VIRUS AND DEVELOPMENT OF RAPID CELL TEST FOR ANTIVIRAL ANTIBODIES ABRAHAM KARPAS P. C. BEVAN
WENDY GILLSON
JOHN K. OATES Department of Haematological Medicine, Cambridge University Clinical School, Hills Road, Cambridge; Department of Genitourinary Medicine, Addenbrooke’s Hospital, Cambridge; and Department of Genitourinary Medicine, Westminster Hospital, London THE acquired immunodeficiency syndrome (AIDS) is caused by a breakdown in the immune response attributed to depletion of helper T-cells, which are killed by a human virus! that is probably of African origin.2 The major mode of viral transmission, apart from homosexual activity, is through blood and its products. It is therefore important to identify potential blood donors who are infected with the virus. The only practical way to do this is to screen for circulating antibodies to the virus. Virus-based enzyme-linked immunosorbent assays (ELISA) give an unacceptable level of false results,3-5 so we have developed a simple and accurate cell test system (Karpas AIDS test) to detect antiviral antibodies, with the use of a local isolate of the AIDS virus replicating in a Karpas T-cell line. The virus was obtained from a practising homosexual in whom AIDS developed; we have reported the expression of unusual virus particles in his cultured cells that were distinct from human T-leukaemic virus type I (adult T-cell leukaemia virus).6 The AIDS virus-infected and non-infected T-cells were used in our virological, serological, and electron microscopy (EM) studies. EM showed a range of morphologically distinct virus-like particles some of which resembled the unusual particles that we initially described in the primary monocytoid cultures from the patient. They were clearly distinct from the enveloped forms but appeared to be developed and associated with the same cells that gave rise to the enveloped forms. In addition to these extracellular forms of virus the infected cells contained large membrane-bound inclusions which were packed with virus
particles (fig 1).
Lytrc Effect of Virus Unlike other viral isolates in other human T-cell lines, the Cambridge isolate of lymphadenopathy AIDS virus (C-LAV) causes a complete cytolysis of the Karpas T-cells. However, since our
Fig 1-Ultra-thin section of part of cytoplasm of C-LAV infected Karpas T-cells showing a membrane-bound inclusion body which is packed with virus particles (rod = 200 nm). T-cell line grows very well in vitro to practically unlimited quantities, a continuous supply of virus-infected cells can be
generated. The Screening Test
System Approximately 105 cells
are
placed
in each well of a multi-well
slide, air dried, and fixed for 10 min in acetone at room temperature. The slides contain three rows of wells. A drop of the test serum or plasma sample diluted 1 in 10 in saline or phosphate-buffered saline (PBS) is placed in each of two wells that contain virus-infected cells and in one well that contains non-infected cells (fig 2). The preparation is incubated in a humidified chamber for 40-60 min at 370C then washed for 10 min in saline or PBS. A suspension of goat antibodies to human immunoglobulin (Sigma) tagged with horse radish peroxidase (HRP) is then added. In initial studies a parallel set of slides was treated with similar goat antibodies tagged with fluorescein. Thus, each serum/plasma sample was tested for antibodies in duplicate and by two methods. Each also included a control. Incubation in a humidified chamber was repeated and followed by washing for 10 min in saline or PBS. Fluoresceinstained preparations were examined with a fluorescence microscope while HRP-treated cells were stained with aminoethyl carbazole or equivalent substrates. The results of the immunoperoxidase (IP) reaction could be clearly distinguished by the naked eye and verified by examination under conventional low-power microscopy. The
696 TABLE 1-RESULTS OF SEROLOGICAL SCREENING
TABLE II-SUMMARY OF THE SEROLOGICAL
TESTS*
*A total of 190 sera from Addenbrooke’s Hospital, Cambridge, and the Westminster Hospital, London, were tested independently both in Cambridge and at the Public Health Laboratory Service (PHLS), London.
Fig 2-Phase
contrast
of IP stained cells
(aminoethyl carbazole).
(A) virus-negative cells show no colour, and (B) virus-positive cells stain a reddish colour which can be seen with the naked eye. Both preparations were incubated with serum containing anti-LAB’ antibodies. immunofluorescence (IF) and IP tests gave the same results. TableI shows the striking difference in the incidence of LAV infection between London and Cambridge homosexuals tested. Table 11 shows the results with 190 sera, which were also tested independently at the Public Health Laboratory Service, London, using the competitive radioimmunoassay (CRIA).7 Unlike the high rate (12/190) of equivocal readings given with CRIA, clear and unambiguous results are given by our IP test. NeutralisatlOn Tests In a preliminary study we tested sera-positive individuals for neutralising antibodies to the AIDS virus. After 1 h incubation of 0’ 1 ml of serum samples with 104 infectious C-LAV particles the Karpas T-cells were infected with the serum/virus mixture. Of the nine sera tested, we found that the two sera from AIDS patients did not block the lytic effect of the virus, whereas five out of seven sera from healthy antibody-positive homosexuals blocked the lytic effect for over 10 days.
The lytic effect of the AIDS virus on the Karpas human T-cells enables us to test for neutralising antibodies also. The high levels of viral antigen which accumulate in the cells make it possible to monitor the results of IP staining with the naked eye. The incorporation in the test slides of wells with virus-negative cells allows the specificity of the reaction for each serum sample to be controlled separately. This ability to discriminate between antiviral and anticellular antibodies is very important in the case of homosexuals and recipients of blood transfusions, who commonly have some anticellular antibodies. Initially we used the IF test in parallel with the IP method, but since our current IP method using staphylococcus-A protein conjugated to HRP (Zymed) gives the same results as the IF test we now use only the IP method, which is simpler. The detection of positive reactions with the naked eye and confirmation with microscopy allows the elimination of the false readings associated with radioactivity or ELISA counters. No specialised expertise is required, nor any sophisticated equipment; a refrigerator and a conventional microscope are all that are needed. Any questionable result can easily be repeated, and in the unlikely event that IP remains questionable, similar slides can also be used in IF tests. The acetone fixation of the slides kills the virus and consequently the slides are entirely safe to handle. We have used satisfactorily slides which had been stored at +4°C for over three months. The Karpas AIDS cell test is a simple and inexpensive method for large-scale screening of blood for anti-LAV antibodies and can give accurate results within 2 h. Moreover, positive sera can be further monitored for neutralising antibodies to the virus with the simple procedure described above, thus, sera with high titres of neutralising antibodies can be selected for possible immunotherapy trials in AIDS patients.
Addendum The donated plasma has been pooled and is being used in our immunotherapy trial of AIDS patients.
Correspondence should be addressed to A. K., Department of Haematological Medicine, University of Cambridge Climcal School, Hills Road, Cambridge CB2 2QL. REFERENCES
Comment The restricted mode of transmission of the AIDS virus should enable effective screening of carriers to check virus spread in the population. Since the commercially available virus-based ELISA test may give misleading results3-) our cell test system, which is simple and appears to be accurate, could provide the method of choice for large-scale screening.
F, Charman JC, Rey F, et al Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS) Science 1983, 220: 868-71 2 Ellrodt A, Barré-Sinoussi F, Le Bras PG, et al Isolation of human T-lymphotropic retrovirus(LAV) from Zairian married couple, one with AIDS, one with prodromes. Lancet 1984; i: 1383-85 1 Barré-Sinoussi
The trial reported here was a pilot study to find out whether a combination of mefloquine with other anti-malarials is as efficient as expected of a long-acting schizonticide to which widespread resistance has not yet developed. Combining mefloquine withSP has been said to delay emergence of resistant strains in P berghei isolates that are chloroquinesensitive or partially resistant to either mefloquine5 or SP. It thus seems sensible for mefloquine to be used only in combination in countries such as Burmawhere a proportion of P falciparum may still be sensitive to SP, so as to prolong the operational life of this drug to a maximum. Another advantage of giving mefloquine in combination is that a smaller dose of the drug will suffice, for therapy or prophylaxis. Cost is extremely important when large populations require a drug, especially an expensive one such as
mefloquine.
SP that was twice that taken before the trial was probably because exposure to malaria was higher during than before the trial. Our findings support the view that MSP should be used for prophylaxis in special high-risk groups continually exposed to multidrug-resistant falciparum malaria. The other drugs now available are so inefficient that their continued use for such groups can no longer be justified. This study was conducted with permission and support from the Directorate of Medical Services, Ministry of Defence. Mefloqume for this study was supplied by Mepha Pharmaceutical Specialties, Basle, Switzerland. Correspondence should be addressed to K. W. REFERENCES
M, Campbell CC, Freeman J, Doberstyn EB, Brandling-Bennett AD. Drug therapy for Plasmodium falciparum malaria resistant to pyrimethamine-sulfadoxine (Fansidar). Lancet 1981, ii: 1066. Hurwitz ES, Johnson D, Campbell CC. Resistance of Plasmodium falciparum malaria
1. Reacher
2.
The group that received MSP became almost completely free from falciparum malaria, whereas in those who received SP the blood film positivity rates for falciparum malaria were high-43’ 66% in group 1 and 70, 37% in group 2. The fact that their positivity rates rose during the trial despite a dose of
to
sulfadoxine-pyrimethamine (Fansidar)
in
a
refugee
camp
in
Thailand. Lancet
1981; i 1068-70. 3. 4 5.
Report of the Fourth Scientific Working Group on the Chemotherapy of Malaria (CHEMAL) WHO Tech Rep Ser no 711, 1984. Peters W. Antimalarial drug resistance. Br Med Bull 1982; 38: 187-92 Report of the Steering Committees ofthe Scientific Working Groups on Malaria. June, 1980-June, 1983. TDR/MAL/SC-SWG (80-83)/83.3.
Methods and Devices LYTIC INFECTION BY BRITISH AIDS VIRUS AND DEVELOPMENT OF RAPID CELL TEST FOR ANTIVIRAL ANTIBODIES ABRAHAM KARPAS P. C. BEVAN
WENDY GILLSON
JOHN K. OATES Department of Haematological Medicine, Cambridge University Clinical School, Hills Road, Cambridge; Department of Genitourinary Medicine, Addenbrooke’s Hospital, Cambridge; and Department of Genitourinary Medicine, Westminster Hospital, London THE acquired immunodeficiency syndrome (AIDS) is caused by a breakdown in the immune response attributed to depletion of helper T-cells, which are killed by a human virus! that is probably of African origin.2 The major mode of viral transmission, apart from homosexual activity, is through blood and its products. It is therefore important to identify potential blood donors who are infected with the virus. The only practical way to do this is to screen for circulating antibodies to the virus. Virus-based enzyme-linked immunosorbent assays (ELISA) give an unacceptable level of false results,3-5 so we have developed a simple and accurate cell test system (Karpas AIDS test) to detect antiviral antibodies, with the use of a local isolate of the AIDS virus replicating in a Karpas T-cell line. The virus was obtained from a practising homosexual in whom AIDS developed; we have reported the expression of unusual virus particles in his cultured cells that were distinct from human T-leukaemic virus type I (adult T-cell leukaemia virus).6 The AIDS virus-infected and non-infected T-cells were used in our virological, serological, and electron microscopy (EM) studies. EM showed a range of morphologically distinct virus-like particles some of which resembled the unusual particles that we initially described in the primary monocytoid cultures from the patient. They were clearly distinct from the enveloped forms but appeared to be developed and associated with the same cells that gave rise to the enveloped forms. In addition to these extracellular forms of virus the infected cells contained large membrane-bound inclusions which were packed with virus
particles (fig 1).
Lytrc Effect of Virus Unlike other viral isolates in other human T-cell lines, the Cambridge isolate of lymphadenopathy AIDS virus (C-LAV) causes a complete cytolysis of the Karpas T-cells. However, since our
Fig 1-Ultra-thin section of part of cytoplasm of C-LAV infected Karpas T-cells showing a membrane-bound inclusion body which is packed with virus particles (rod = 200 nm). T-cell line grows very well in vitro to practically unlimited quantities, a continuous supply of virus-infected cells can be
generated. The Screening Test
System Approximately 105 cells
are
placed
in each well of a multi-well
slide, air dried, and fixed for 10 min in acetone at room temperature. The slides contain three rows of wells. A drop of the test serum or plasma sample diluted 1 in 10 in saline or phosphate-buffered saline (PBS) is placed in each of two wells that contain virus-infected cells and in one well that contains non-infected cells (fig 2). The preparation is incubated in a humidified chamber for 40-60 min at 370C then washed for 10 min in saline or PBS. A suspension of goat antibodies to human immunoglobulin (Sigma) tagged with horse radish peroxidase (HRP) is then added. In initial studies a parallel set of slides was treated with similar goat antibodies tagged with fluorescein. Thus, each serum/plasma sample was tested for antibodies in duplicate and by two methods. Each also included a control. Incubation in a humidified chamber was repeated and followed by washing for 10 min in saline or PBS. Fluoresceinstained preparations were examined with a fluorescence microscope while HRP-treated cells were stained with aminoethyl carbazole or equivalent substrates. The results of the immunoperoxidase (IP) reaction could be clearly distinguished by the naked eye and verified by examination under conventional low-power microscopy. The
696 TABLE 1-RESULTS OF SEROLOGICAL SCREENING
TABLE II-SUMMARY OF THE SEROLOGICAL
TESTS*
*A total of 190 sera from Addenbrooke’s Hospital, Cambridge, and the Westminster Hospital, London, were tested independently both in Cambridge and at the Public Health Laboratory Service (PHLS), London.
Fig 2-Phase
contrast
of IP stained cells
(aminoethyl carbazole).
(A) virus-negative cells show no colour, and (B) virus-positive cells stain a reddish colour which can be seen with the naked eye. Both preparations were incubated with serum containing anti-LAB’ antibodies. immunofluorescence (IF) and IP tests gave the same results. TableI shows the striking difference in the incidence of LAV infection between London and Cambridge homosexuals tested. Table 11 shows the results with 190 sera, which were also tested independently at the Public Health Laboratory Service, London, using the competitive radioimmunoassay (CRIA).7 Unlike the high rate (12/190) of equivocal readings given with CRIA, clear and unambiguous results are given by our IP test. NeutralisatlOn Tests In a preliminary study we tested sera-positive individuals for neutralising antibodies to the AIDS virus. After 1 h incubation of 0’ 1 ml of serum samples with 104 infectious C-LAV particles the Karpas T-cells were infected with the serum/virus mixture. Of the nine sera tested, we found that the two sera from AIDS patients did not block the lytic effect of the virus, whereas five out of seven sera from healthy antibody-positive homosexuals blocked the lytic effect for over 10 days.
The lytic effect of the AIDS virus on the Karpas human T-cells enables us to test for neutralising antibodies also. The high levels of viral antigen which accumulate in the cells make it possible to monitor the results of IP staining with the naked eye. The incorporation in the test slides of wells with virus-negative cells allows the specificity of the reaction for each serum sample to be controlled separately. This ability to discriminate between antiviral and anticellular antibodies is very important in the case of homosexuals and recipients of blood transfusions, who commonly have some anticellular antibodies. Initially we used the IF test in parallel with the IP method, but since our current IP method using staphylococcus-A protein conjugated to HRP (Zymed) gives the same results as the IF test we now use only the IP method, which is simpler. The detection of positive reactions with the naked eye and confirmation with microscopy allows the elimination of the false readings associated with radioactivity or ELISA counters. No specialised expertise is required, nor any sophisticated equipment; a refrigerator and a conventional microscope are all that are needed. Any questionable result can easily be repeated, and in the unlikely event that IP remains questionable, similar slides can also be used in IF tests. The acetone fixation of the slides kills the virus and consequently the slides are entirely safe to handle. We have used satisfactorily slides which had been stored at +4°C for over three months. The Karpas AIDS cell test is a simple and inexpensive method for large-scale screening of blood for anti-LAV antibodies and can give accurate results within 2 h. Moreover, positive sera can be further monitored for neutralising antibodies to the virus with the simple procedure described above, thus, sera with high titres of neutralising antibodies can be selected for possible immunotherapy trials in AIDS patients.
Addendum The donated plasma has been pooled and is being used in our immunotherapy trial of AIDS patients.
Correspondence should be addressed to A. K., Department of Haematological Medicine, University of Cambridge Climcal School, Hills Road, Cambridge CB2 2QL. REFERENCES
Comment The restricted mode of transmission of the AIDS virus should enable effective screening of carriers to check virus spread in the population. Since the commercially available virus-based ELISA test may give misleading results3-) our cell test system, which is simple and appears to be accurate, could provide the method of choice for large-scale screening.
F, Charman JC, Rey F, et al Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS) Science 1983, 220: 868-71 2 Ellrodt A, Barré-Sinoussi F, Le Bras PG, et al Isolation of human T-lymphotropic retrovirus(LAV) from Zairian married couple, one with AIDS, one with prodromes. Lancet 1984; i: 1383-85 1 Barré-Sinoussi