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Antigen testing in HIV-1 infection
Serodiagnosis
and Immunotherapy
in Infectious Disease (1988) 2, 233-240
Antigen testing in HIV-l
infection
Donald J. Jeffries St. Mary’s Hospital
Medical
School, Paddington,
London
W2 IPG, U.K.
Introduction The acquired immune deficiency syndrome (AIDS) was first reported in the United States in 1981’ and its association with infection by the human immunodeficiency virus type 1 (HIV-l) was shown in 1983-842,3. Following the adaptation of HIV-1 to a continuously growing line of human T-lymphoblastoid cells4, various serologic tests have been developed to detect the presence of antibodies to HIV-l in the sera of asymptomatic carriers of the virus and in patients with AIDS and AIDS-related conditions (ARC)‘. These antibody tests include enzyme-linked immunosorbent assays(ELISA), radioimmune precipitation assays(RIPA), immunofluorescence and immunoblotting (Western blotting) procedures. Genomic sequencing has led to the availability of recombinant HIV-l proteins, and this has allowed the development of ELISA techniques for the detection and quantitation of specific antibodies to HIV-l core (~24) and envelope (gp41) proteins in the sera of plasma of infected individuals6,7. Increasingly sensitive antibody screening tests have improved the detection of carriers of HIV- 1, and study of levels of specific antibodies at different stages of the disease process has revealed a correlation between the decline of antibody to the p24 protein8,9, and possibly ~17”). and progression to full-blown AIDS. In order to investigate the activity of the virus in an infected individual there was a need for a convenient assay for virus production or expression of viral antigens. The presence of integrated provirus in the circulating mononuclear cells of asymptomatic carriers and patients with HIV-l related diseases means that induction of virus production from cultured cells cannot be indicative of the activity of these cells in viva. HIV-l can be isolated from plasma samplesearly in the infectious process” but this technique is unsuitable for the routine assessmentof infected individuals. The development of a sensitive assay for HIV-l antigen “,‘3 has proved to be a valuable indicator of virus expression during the course of infection. The majority of published work, in which HIV-l antigen responsesare characterized, is basedon a solid phase, sandwich-type enzyme immunoassay (Abbott Laboratories) in which human and rabbit polyclonal anti-HIV-l IgG are used as capture and probe antibodies respectively. Patients’ sera (200 ~1) are incubated overnight at room tempera233 08884786/88/040233
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D. J. Jeffries
ture with polystyrene beads coated with human anti-HIV-l IgG. After washing, rabbit antibodies to HIV- 1 (predominantly anti-p24) are added and the beads are incubated at 40°C for 4 h. After further washing, the next step involves the addition of goat-antirabbit IgG labelled with horseradish peroxidase. Colour is developed with orthophenylenediamine as substrate, and absorbance is read at 492 nm. Results are recorded as positive if the optical density is > 0.050 plus the mean of five replicates of normal human plasma. The level of antigen can be quantified by plotting unknown positive samples against a reference curve obtained by testing serial dilutions of purified HIV-l lysate. The power limit of detection in these assays is 20-30 pg ml’. False positives may occur in this test13,14and a neutralization, or blocking, test should be performed on repeatedly positive samples to verify the test results. HIV-l antigen in the sample is neutralized by incubating 200 ul of serum with 50 ul of a polyclonal human anti-HIV-l IgG solution prior to running the antigen assay”. A reduction of at least 50% in the optical density reading may be taken as confirmation of a positive HIV-l antigen result. The development of this sensitive assay for HIV-l antigenaemia has made a major contribution to our understanding of the infectious process. There is, however, a need for further development as the prolonged incubation periods render the test unsuitable if it is to be considered for donor screening. In addition to this, the relatively large volumes of serum necessary to conduct the initial test and the confirmatory and neutralization tests cause problems in certain clinical situations such as the testing of babies and samples of cerebrospinal fluid. At the time of writing, other manufacturers are introducing antigen tests, and some of these incorporate shorter incubation times and smaller volumes of serum. Further assessment is necessary to determine whether these assays are as sensitive and specific as the method outlined above.
Early antigenaemia In studies of sequential serum samples from people infected with HIV-l, several groups have described a period of antigenaemia prior to the development of detectable antibody. This was first described in a homosexual man by Lange et ~1.‘~ and in haemophiliacs by Allain et al.13. Following the demonstration of marked plasma viraemia, by virus isolations, during the early phase of primary infection with HIV- 1‘i , Gaines et al.” showed that the ability to culture virus was correlated with detectable antigenaemia. Von Sydow et a/.‘* obtained serial serum samples from 21 male homosexuals with clinical illness associated with seroconversion to HIV-l. In nine of these patients the date of infection could be determined with reasonable certainty and the incubation period prior to illness had a median duration of 14 days (range 1 l-28 days). During the first week of illness, p24 antigen was detectable in the absence of antibodies. In the second and third weeks after onset of the illness, p24 antibody became detectable by Western blotting and antigen levels decreased rapidly to undetectable levels. During this phase of declining antigenaemia, dissociation of circulating immune complexes revealed the presence of complexed p24 antigen. The fact that neither free nor complexed p24 antigen could be detected in any serum samples for several months thereafter may provide some support to the concept of a period of latent infection following seroconversion. However, Gaines et a1.7 had demonstrated previously that serum may contain infectious virus in the absence of detectable antigenaemia.
Antigen testing in HIV-1 infection
235
The association of an antibody-negative, antigenaemic phase with primary symptomatic HIV-l infection was also described in four homosexual men by Kessler et ~1.‘~. In studies of haemophiliacs in Edinburgh, infected from a single batch of contaminated factor VIII during March 1984, Simmonds et ~1.” reported variable time periods to seroconversion and one patient remained antibody-negative for at least 160 days. Antigenaemia was present in five of 12 patients for whom pre-seroconversion sera were available, and none of the infected patients appear to have suffered a primary seroconversion illness. The fact that there is a well-documented phase of antigenaemia prior to seroconversion has caused anxiety over the safety of blood for transfusion and tissues for transplantation. In the United Kingdom, the incidence of HIV-l seropositivity in blood donors, after voluntary exclusion of those exposed to high-risk activity, has been remarkably low (approximately I in 50,000) and the danger of a donor being antibodynegative, antigen-positive has been estimated as less than one in one million donations. Against this minute risk must be balanced the much higher risk of severe transfusion reactions and the cost-effectiveness of introducing antigen testing at this time. At present, the high cost of reagents and the difficulties presented by a test that takes 24 hours are major considerations in the decision. As refinement of antibody tests occurs, their sensitivity is increasing and positivity is detectable increasingly early in the post-infection period. This is continually reducing the duration of the period of seronegative antigenaemia and is thus decreasing the transfusion hazard. If the seroprevalence of HIV-I reaches the levels in blood donors which will necessitate routine antigen screening it is likely that newer, less prolonged and less costly tests will be available for blood and organ donor screening. Late antigenaemia
Sequential studies of antibodies in cohorts of HIV-l infected individuals have shown that declining levels of antibodies to core proteins, particularly anti-p24, may precede the development of AIDS by many months*‘***. Subsequent studies have demonstrated the development of persistent antigenaemia which follows the p24 antibody decline and also shows a statistically significant correlation with disease progression. In studies of HIV-l infection in homosexual men in Amsterdam, de Wolf et al. reported that the incidence of AIDS was some 20 times higher in seropositive men who had antigenaemia than in those who were antigen negative 23. In a 3-year actuarial analysis of 288 seropositive men enlisted into a cohort study in San Fransisco, the relative prognostic importance of a number of clinical and laboratory parameters was assessed, singly and in combination”. It was estimated that the cohort were infected approximately 3 years before enrolment. Progression to AIDS over 3 years was estimated as 59% in seropositive individuals who were p24-antigenaemic at enrolment, compared with 15% in those who were antigen negative. Allain et ~1.~confirmed the predictive value of serum p24 antigen detection in patients with haemophilia. The apparent reciprocal relationship between the rise and persistence of p24-antigenaemia and the decline of p24 antibodies, which has been ascribed by Lange et uf.lh to immune complex formation, is not always observed. In follow-up of the Edinburgh haemophiliac cohort, Simmonds et al. described one patient in whom p24 antibody returned from an undetectable level in the face of increasing amounts of antigen’“.
236
D. J. Jeffries
Despite the strong association of AIDS with p24 antigenaemia there is a significant proportion of AIDS patients who remain antigen negative; in the cohort studies of Kenny et al. 70% of AIDS patients were antigen positive14. Similarly, although loss of p24 antibody and development of p24 antigenaemia are both highly predictive of disease, some patients remain disease-free for years despite these findings. Thus the use of these tests must be introduced with caution in the assessment of HIV-l seropositive individuals. The pattern of responses in African patients is quite different. Baillou et al.*’ found only one of 40 patients with AIDS in Burundi were antigenaemic and another 40 with ARC were negative. These findings were associated with high and persisting levels of p24 antibody in the African patients. Although some have suggested that persistence of p24 antibody may play a role in protecting against the onset of AIDS, Baillou ef al. stress that this was not the case in these patients who progressed to disease despite the presence of antibody. The demonstration that development of antigenaemia in HIV- 1 infected individuals is significantly associated with progression to AIDS is of major importance in selecting patients for trials of anti-viral agents. If this, and other, predictive assessments indicate that an individual who is currently well, or has only minor symptoms, is likely to suffer rapid deterioration in the coming months, an effective anti-retroviral drug may be highly effective if administered at this time. This is particularly likely if the immune system has not been too severely damaged by the virus. In addition to this, the presence of p24 antigenaemia offers a parameter for assessing antiviral activity of drugs in viva. Trials of anti-retroviral
drugs
A placebo-controlled trial of zidovudine (3’-azido-3’-deoxythymidine, azidothymidine, AZT) treatment for patients with AIDS and severe ARC provided the opportunity to study various features of infection with HIV-I 26. The placebo group was discontinued and offered zidovudine when it became clear that significantly more were surviving in the zidovudine-treated group. Jackson et al.*’ reported serial p24 antigen levels in 12 patients who had, or developed, antigenaemia ranging from 16 to 3006 pgml-‘. Zidovudine, 200 or 250 mg every 4 hours, reduced antigenaemia by approximately 90%; other dosage regimens were less effective. Although it was still possible to isolate virus from cultured lymphocytes during full dose zidovudine therapy this, and subsequent, studies** have shown a correlation between clinical benefit and an antiviral effect. de Wolf et a1.29 measured the effect of zidovudine on serum p24 antigen levels in 18 symptom-free men with longstanding HIV-l antigenaemia. The antigen level rose in only one patient and declined significantly in 13 (to below cut-off levels in nine). A virological effect, as judged by disappearance of circulating p24 antigen, was observed in patients with AIDS receiving foscarnet (phosphonoformate) by continuous infusion30, and some effect on antigenaemia was claimed in phase 1 studies of 2’ 3’dideoxycytidine 31. The lack of clinical response to suramin in patients with AIDS and ARC reported by Kaplan et aL3’ was supported by an absence of virological effect in a report from another group”. As none of these putative reverse transcriptase inhibitors can be expected to influence the existence of an integrated provirus in lymphocytes or other cells which are already infected, virus culture has little, if anything, to offer in their assessment in viva. As has been shown from studies conducted to date, if patients have pre-existing antigenaemia, monitoring of antigen levels during therapy may be used to assessactivity of the virus. It
Antigen testing in HIV-l
infection
237
is likely, therefore, that as new drugs with acceptably low toxicity profiles are developed. their toxicity in humans and antiviral efficacy will be determined by phase 1 trials in small groups of HIV-l infected volunteers who are p24 antigen positive. Detection of infection in infants
There are problems in the diagnosis of HIV-l infection in the offspring of women who are seropositive. Persistence of maternal antibody, which may be detected for over one year by currently available ELISAs, may mask low levels of antibody production in the child, and there is no satisfactory specific IgM test as yet. In addition, virus isolation has been achieved from some infants who are totally negative by all antibody testing systems. Borkowsky et ~1.‘~ reported the finding of HIV- 1 antigenaemia in 9/58 children, with clinical, epidemiological or immunological evidence of infection, all of whom had negative antibody ELISA results. In a further report, these workers found p24 antigenaemia in 53% of asymptomatic children born to HIV-l antibody positive women, 70% of children with ARC and 85% of children who had experienced an opportunistic infection 35. It remains to be seen, however. whether antigen testing will be a useful addition to virus culture and sequential antibody measurement in assessment of infection in neonates and children. HIV-l
antigen in body fluids other than blood
HIV-l antigen can be detected in the cerebrospinal fluid (CSF) of some HIV-1 positive patients. Goudsmit et al.” reported positivity in all 5 children and S/9 adults with AIDSrelated encephalopathy, and negative antigen tests in 13 seropositive controls. However, longitudinal study of one of the controls showed that HIV-l antigen had been present transiently before the development of antibody in the CSF. These workers suggested that the persistence or reappearance of antigen in CSF may correlate with neurological deterioration, Others have confirmed the biphasic nature of CSF antigen positivity with an early transient rise followed by later persistence in some patients36. Rasheed et al. detected found p24 antigen in 5/7 (71%) of patients with encephalitis and 4/24 (16%) of patients without signs of central nervous system disease”; they pointed out, however, that the assay could not distinguish between encephalitis due to toxoplasmosis and that caused solely by HIV- I. It remains to be determined whether the presence and quantity of antigen in CSF can be used to assess the extent and prognosis of neurological damage by HIV-I and, possibly, intracranial opportunist infections. In the course of treating patients with AIDS and ARC with low-dose zidovudine (250 mg, 6-hourly), de Gans et ~1.~’ reported disappearance of HIV-1 antigen from CSF within 8 weeks in three patients who had previously been persistently positive. This occurred in the absence of significant neurological improvement. Interruption of therapy in one patient led to the reappearance of antigen which reached a high level and then disappeared when zidovudine was restarted. HIV- I antigen testing has been applied to other body fluids including saliva, bronchoalveolar lavage and semen in attempts to assess the level of virus excretion. Unfortunately the finding of a high degree of false positivity has hampered this work and it remains to be seen whether the test can be used for this purpose. Reports of positivity in broncho-alveolar lavage in lymphocytic interstitial pneumonitis” and not in other AIDS-related lung diseases require further investigation.
238
D. J. Jeffriea Conclusions
The introduction of antigen tests for HIV-l infection has provided a valuable contribution to diagnosis of infection and an important predictive marker of disease progression. The occurrence of false-positivity, albeit at a low level in serum and plasma samples, necessitates the use of a neutralization or blocking test. The use of the test for determining prognosis must be handled with care, as some individuals can remain antigen positive and asymptomatic for years, but it may prove to be of value in selecting patients for trials and clinical use of new anti-retroviral agents as they become available. The presence of antigen in blood and CSF can be used as a marker for assessing the efficacy of anti-retroviral agents in vim, and, as far as can be judged to date, depression of antigenaemia has correlated with clinical improvement. As in other areas of AIDS research, technological advances are occurring rapidly and new tests, detecting antigens other than ~24, requiring smaller sample volumes, and having shorter incubation times and greater sensitivity levels, should soon be evaluated.
References
I. Centers for Disease Control (CDC). Kaposi’s sarcoma and pneumocystis pneumonia among homosexualmen-New York City and California. MMWR 1981;30: 305-8. 2. Barre-SinoussiF, ChermannJ-C, Rey F et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquiredimmunedeficiency syndrome(AIDS). Science1983;220: 868-71. 3 Gallo RC, SalahuddinSZ, Popovic M et al. Frequent detection and isolation of cytopathic retroviruses(HTLV-111) from patientswith AIDS and at risk from AIDS. Science1984;224: 500-3. 4. Popovic M, SarngadharanM, Read E et al. Detection, isolation and continuousproduction of cytopathic retroviruses(HTLV-111) from patientswith AIDS and pre-AIDS. Science1984; 224: 497-500. 5. Mortimer PP, Clewley JP. Serologicaltestsfor humanimmunodeficiencyvirus. In: Gottleib MS, JeffriesDJ, Mildvan D et al., eds.Current Topicsin AIDS. Chichester:John Wiley, 1987: 133-54. 6. Allain J-P, Laurian Y, Paul DA et at. Long-termevaluation of HIV antigenand antibodiesto p24 and gp41 in patientswith haemophilia.N Eng J Med 1987;317: 111421. 7. Goudsmit J, Lange JMA, Paul DA, Dawson GJ. Antigenaemiaand antibody titres to core and envelope antigensin AIDS, AIDS-related complex, and subclinicalhuman immunodeficiencyvirus infection. J Infect Dis 1987;155:558-60. 8. LangeJMA, Paul DA, HuismanHG et al. PersistentHIV antigenaemiaand declineof HIV core antibodiesassociatedwith transition to AIDS. Br Med J 1986;293: 1459-62. 9. Paul DA, Falk LA, KesslerHA et al. Correlation of serumHIV antigen and antibody with clinical statusin HIV-infected patients.J Med Virol 1987;22: 357-63. 10. LangeJMA. de Wolf F, Krone WJA et al. Declineof antibody reactivity to outer virual core protein pl7 is an earlier serologicalmarker of diseaseprogressionin human immunodeficiencyvirus infection than anti-p4 decline.AIDS 1987;1: 155-9. I I. Albert J, GainesH, SonnerbergA et al. Isolation of human immunodeficiencyvirus (HIV) from plasmaduring primary HIV infection. J Med Virol 1987;23: 67-73. 12. Goudsmit J, Paul DA, Lange JMA et al. Expressionof human immunodeficiencyvirus antigen(HIV-Ag) in serumand cerebrospinalfluid during acuteand chronic infection. Lancet 1986;i: 177-80. 13. Allain JP, Laurian Y, Paul DA et al. Serological markers in early stagesof human immunodeficiencyvirus infection in haemophiliacs.Lancet 1986;ii: 1233-6. 14. Kenny C, Parkin J, Underhill G et al. HIV antigen testing. Lancet 1987;i: 565-6. 15. Cao Y, Valentine F, Hojvat S et al. Detection of HIV antigen and specificantibodiesto HIV core and envelopeproteins in seraof patientswith HIV infection. Blood 1987;70: 575-8.
Antigen testing in HIV-1
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16. Lange JMA, Paul DA, de Wolf F et al. Viral geneexpression,antibody production and immunecomplex formation in humanimmunodeficiencyvirus infection. AIDS 1987; 1: 15.20.
17. GainesH, Albert J, von Sydow M et al. HIV antigenaemiaand virus isolation from plasma during primary HIV infection. Lancet 1987;i: 1317-8. 18. von Sydow M, GainesH, SonnerborgA et al. Antigen detectionin primary HIV infection. Br Med J 1988;296: 238-240. 19. KesslerHH, BlaauwB, SpearJ et al. Diagnosisof humanimmunodeficiencyvirus infection in seronegativehomosexualspresentingwith an acuteviral syndrome.JAMA 1987;258: 1196-9. 20. SimmondsP, Lainson FAL, Cuthbert R et al. HIV antigenand antibody detection: variable responses to infection in the Edinburgh haemophiliaccohort. Br Med J 1988;296: 593-8. 21, Weber JN, ClaphamPR, WeissRA et al. Human immunodeficiencyvirus infection in two cohorts of homosexualmen: neutralizing sera and associationof anti-gag antibody with prognosis.Lancet 1987;i: 119-22. 22. Forster SM, OsborneLM, Cheingsong-PopovR et al. Declineof anti-p24 antibody precedes antigenaemiaascorrelate of prognosisin HIV-1 infection, AIDS 1987;1: 23540. 23. de Wolf F, Goudsmit J, Paul DA et al. Risk of AIDS related complex and AIDS in homosexualmenwith persistentHIV antigenaemia.BMJ 1987;295: 569-72. 34. Moss AR, BacchettiP, OsmondD et al. Seropositivity for HIV and the developmentof AIDS or AIDS related condition: three year follow up of the San Francisco General Hospital cohort. Br Med J 1988;296: 745-50. 25. Baillou A, Barin F, Allain JP et al. Human immunmodeficiencyvirus antigenaemiain patients with AIDS and AIDS-related disorders:a comparisonbetweenEuropeanand Central African populations. J Infect Dis 1987;156:830-2. 26. Fischl M, Grieco MH, Richman D et al. The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex: a double-blind placebocontrolled trial. N Eng J Med 1987;317: 185-91. 27. JacksonGG, Paul DA, Falk LA et al. Human immunodeficiencyvirus (HIV) antigenaemia (~24) in the acquired immunodeficiencysyndrome(AIDS) and the effect of treatment with zidovudine (AZT). Ann Int Med 1988;108: 175-80. 28. Helbert M, PeddleB, RobinsonD et al. Clinical experiencewith zidovudine in the treatment of patientswith AIDS and AIDS-related complex. In preparation. 29. de Wolf F, Goudsmit J, de Gans J et al. Effect of zidovudine on serumhuman immunodeficiencyvirus antigenlevelsin symptom-freesubjects.Lancet 1988;i: 373-5. 30. Gaub J, PedersenC, PoulsenA-G et al. The effect of foscarnet(phosphonoformate)on human immunodeficiencyvirus isolation, T-cell subsetsand lymphocyte function in AIDS patients. AIDS 1987;1: 27-33. 3I Yarchoan R, Perno CF, ThomasRV et al. Phase1 studiesof 2’,3’-dideoxycytidine in severe human immunodeficiencyvirus infection as a singleagent and alternating with zidovudine (AZT). Lancet 1988;i: 7681. 32. Kaplan LD, Wolfe PR, Volberding PA et al. Lack of responseto suraminin patientswith AIDS and AIDS-related complex. Am J Med 1987;82: 615-20. 33. SchattenkerkJKME, Danner SA, LangeJMA et al. Persistence of humanimmunodeficiency virus antigenaemiain patientswith the acquiredimmunodeficiencysyndrometreated with a reversetranscriptaseinhibitor, suramin.Arch Int Med 1988;148:209-I I. 34. Borkowsky W, Krasinski K, Paul D et al. Human immunodeficiencyvirus infections in infants negative for anti-HIV by enzyme-Iinkedimmunoassay.Lancet 1987;i: I 168-71. 35. Borkowsky W, Krasinski K, PaulD et al. Humanimmunodeficiencyvirus (HIV) core protein antigenaemiain children with HIV infection. Abstr 7262. IV International Conferenceon Aids, Stockholm 1988. 36. Angarano G, CarbonaraS, CasalinoC et al. Blood-brain barrier damage,HIV p24 antigenin cerebrospinaffluid and intrathecal synthesisof total and HIV-specific IgG on patients with symptomatic or asymptomaticHIV infection. Abstr 1634.IV International Conferenceon AIDS, Stockholm 1988. 37. RasheedS, Su S, LarsenRA. Correlation of HIV p24 levelsin cerebrospinalfluid (CSF) and encephalitisin HIV-infected patients. Abstr 7762. IV International Conferenceon AIDS. Stockholm 1988.
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38. de Gans J, Lange JMA, Derix MMA et al. Decline of HIV antigen levels in cerebrospinal fluid during treatment with low-dose zidovudine. AIDS 1988; 1: 3740. 39. Linnemann CC, Baughman RP, Frame PT, Floyd R. Human immunodeficiency virus and antigen in bronchoalveolar lavege fluid from adult patients with AIDS. Abstr 7136. IV International Conference on AIDS. Stockholm 1988.
and Immunotherapy
in Infectious Disease (1988) 2, 233-240
Antigen testing in HIV-l
infection
Donald J. Jeffries St. Mary’s Hospital
Medical
School, Paddington,
London
W2 IPG, U.K.
Introduction The acquired immune deficiency syndrome (AIDS) was first reported in the United States in 1981’ and its association with infection by the human immunodeficiency virus type 1 (HIV-l) was shown in 1983-842,3. Following the adaptation of HIV-1 to a continuously growing line of human T-lymphoblastoid cells4, various serologic tests have been developed to detect the presence of antibodies to HIV-l in the sera of asymptomatic carriers of the virus and in patients with AIDS and AIDS-related conditions (ARC)‘. These antibody tests include enzyme-linked immunosorbent assays(ELISA), radioimmune precipitation assays(RIPA), immunofluorescence and immunoblotting (Western blotting) procedures. Genomic sequencing has led to the availability of recombinant HIV-l proteins, and this has allowed the development of ELISA techniques for the detection and quantitation of specific antibodies to HIV-l core (~24) and envelope (gp41) proteins in the sera of plasma of infected individuals6,7. Increasingly sensitive antibody screening tests have improved the detection of carriers of HIV- 1, and study of levels of specific antibodies at different stages of the disease process has revealed a correlation between the decline of antibody to the p24 protein8,9, and possibly ~17”). and progression to full-blown AIDS. In order to investigate the activity of the virus in an infected individual there was a need for a convenient assay for virus production or expression of viral antigens. The presence of integrated provirus in the circulating mononuclear cells of asymptomatic carriers and patients with HIV-l related diseases means that induction of virus production from cultured cells cannot be indicative of the activity of these cells in viva. HIV-l can be isolated from plasma samplesearly in the infectious process” but this technique is unsuitable for the routine assessmentof infected individuals. The development of a sensitive assay for HIV-l antigen “,‘3 has proved to be a valuable indicator of virus expression during the course of infection. The majority of published work, in which HIV-l antigen responsesare characterized, is basedon a solid phase, sandwich-type enzyme immunoassay (Abbott Laboratories) in which human and rabbit polyclonal anti-HIV-l IgG are used as capture and probe antibodies respectively. Patients’ sera (200 ~1) are incubated overnight at room tempera233 08884786/88/040233
+ 08 $03.00/O
@J1988AcademicPressLimited
234
D. J. Jeffries
ture with polystyrene beads coated with human anti-HIV-l IgG. After washing, rabbit antibodies to HIV- 1 (predominantly anti-p24) are added and the beads are incubated at 40°C for 4 h. After further washing, the next step involves the addition of goat-antirabbit IgG labelled with horseradish peroxidase. Colour is developed with orthophenylenediamine as substrate, and absorbance is read at 492 nm. Results are recorded as positive if the optical density is > 0.050 plus the mean of five replicates of normal human plasma. The level of antigen can be quantified by plotting unknown positive samples against a reference curve obtained by testing serial dilutions of purified HIV-l lysate. The power limit of detection in these assays is 20-30 pg ml’. False positives may occur in this test13,14and a neutralization, or blocking, test should be performed on repeatedly positive samples to verify the test results. HIV-l antigen in the sample is neutralized by incubating 200 ul of serum with 50 ul of a polyclonal human anti-HIV-l IgG solution prior to running the antigen assay”. A reduction of at least 50% in the optical density reading may be taken as confirmation of a positive HIV-l antigen result. The development of this sensitive assay for HIV-l antigenaemia has made a major contribution to our understanding of the infectious process. There is, however, a need for further development as the prolonged incubation periods render the test unsuitable if it is to be considered for donor screening. In addition to this, the relatively large volumes of serum necessary to conduct the initial test and the confirmatory and neutralization tests cause problems in certain clinical situations such as the testing of babies and samples of cerebrospinal fluid. At the time of writing, other manufacturers are introducing antigen tests, and some of these incorporate shorter incubation times and smaller volumes of serum. Further assessment is necessary to determine whether these assays are as sensitive and specific as the method outlined above.
Early antigenaemia In studies of sequential serum samples from people infected with HIV-l, several groups have described a period of antigenaemia prior to the development of detectable antibody. This was first described in a homosexual man by Lange et ~1.‘~ and in haemophiliacs by Allain et al.13. Following the demonstration of marked plasma viraemia, by virus isolations, during the early phase of primary infection with HIV- 1‘i , Gaines et al.” showed that the ability to culture virus was correlated with detectable antigenaemia. Von Sydow et a/.‘* obtained serial serum samples from 21 male homosexuals with clinical illness associated with seroconversion to HIV-l. In nine of these patients the date of infection could be determined with reasonable certainty and the incubation period prior to illness had a median duration of 14 days (range 1 l-28 days). During the first week of illness, p24 antigen was detectable in the absence of antibodies. In the second and third weeks after onset of the illness, p24 antibody became detectable by Western blotting and antigen levels decreased rapidly to undetectable levels. During this phase of declining antigenaemia, dissociation of circulating immune complexes revealed the presence of complexed p24 antigen. The fact that neither free nor complexed p24 antigen could be detected in any serum samples for several months thereafter may provide some support to the concept of a period of latent infection following seroconversion. However, Gaines et a1.7 had demonstrated previously that serum may contain infectious virus in the absence of detectable antigenaemia.
Antigen testing in HIV-1 infection
235
The association of an antibody-negative, antigenaemic phase with primary symptomatic HIV-l infection was also described in four homosexual men by Kessler et ~1.‘~. In studies of haemophiliacs in Edinburgh, infected from a single batch of contaminated factor VIII during March 1984, Simmonds et ~1.” reported variable time periods to seroconversion and one patient remained antibody-negative for at least 160 days. Antigenaemia was present in five of 12 patients for whom pre-seroconversion sera were available, and none of the infected patients appear to have suffered a primary seroconversion illness. The fact that there is a well-documented phase of antigenaemia prior to seroconversion has caused anxiety over the safety of blood for transfusion and tissues for transplantation. In the United Kingdom, the incidence of HIV-l seropositivity in blood donors, after voluntary exclusion of those exposed to high-risk activity, has been remarkably low (approximately I in 50,000) and the danger of a donor being antibodynegative, antigen-positive has been estimated as less than one in one million donations. Against this minute risk must be balanced the much higher risk of severe transfusion reactions and the cost-effectiveness of introducing antigen testing at this time. At present, the high cost of reagents and the difficulties presented by a test that takes 24 hours are major considerations in the decision. As refinement of antibody tests occurs, their sensitivity is increasing and positivity is detectable increasingly early in the post-infection period. This is continually reducing the duration of the period of seronegative antigenaemia and is thus decreasing the transfusion hazard. If the seroprevalence of HIV-I reaches the levels in blood donors which will necessitate routine antigen screening it is likely that newer, less prolonged and less costly tests will be available for blood and organ donor screening. Late antigenaemia
Sequential studies of antibodies in cohorts of HIV-l infected individuals have shown that declining levels of antibodies to core proteins, particularly anti-p24, may precede the development of AIDS by many months*‘***. Subsequent studies have demonstrated the development of persistent antigenaemia which follows the p24 antibody decline and also shows a statistically significant correlation with disease progression. In studies of HIV-l infection in homosexual men in Amsterdam, de Wolf et al. reported that the incidence of AIDS was some 20 times higher in seropositive men who had antigenaemia than in those who were antigen negative 23. In a 3-year actuarial analysis of 288 seropositive men enlisted into a cohort study in San Fransisco, the relative prognostic importance of a number of clinical and laboratory parameters was assessed, singly and in combination”. It was estimated that the cohort were infected approximately 3 years before enrolment. Progression to AIDS over 3 years was estimated as 59% in seropositive individuals who were p24-antigenaemic at enrolment, compared with 15% in those who were antigen negative. Allain et ~1.~confirmed the predictive value of serum p24 antigen detection in patients with haemophilia. The apparent reciprocal relationship between the rise and persistence of p24-antigenaemia and the decline of p24 antibodies, which has been ascribed by Lange et uf.lh to immune complex formation, is not always observed. In follow-up of the Edinburgh haemophiliac cohort, Simmonds et al. described one patient in whom p24 antibody returned from an undetectable level in the face of increasing amounts of antigen’“.
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D. J. Jeffries
Despite the strong association of AIDS with p24 antigenaemia there is a significant proportion of AIDS patients who remain antigen negative; in the cohort studies of Kenny et al. 70% of AIDS patients were antigen positive14. Similarly, although loss of p24 antibody and development of p24 antigenaemia are both highly predictive of disease, some patients remain disease-free for years despite these findings. Thus the use of these tests must be introduced with caution in the assessment of HIV-l seropositive individuals. The pattern of responses in African patients is quite different. Baillou et al.*’ found only one of 40 patients with AIDS in Burundi were antigenaemic and another 40 with ARC were negative. These findings were associated with high and persisting levels of p24 antibody in the African patients. Although some have suggested that persistence of p24 antibody may play a role in protecting against the onset of AIDS, Baillou ef al. stress that this was not the case in these patients who progressed to disease despite the presence of antibody. The demonstration that development of antigenaemia in HIV- 1 infected individuals is significantly associated with progression to AIDS is of major importance in selecting patients for trials of anti-viral agents. If this, and other, predictive assessments indicate that an individual who is currently well, or has only minor symptoms, is likely to suffer rapid deterioration in the coming months, an effective anti-retroviral drug may be highly effective if administered at this time. This is particularly likely if the immune system has not been too severely damaged by the virus. In addition to this, the presence of p24 antigenaemia offers a parameter for assessing antiviral activity of drugs in viva. Trials of anti-retroviral
drugs
A placebo-controlled trial of zidovudine (3’-azido-3’-deoxythymidine, azidothymidine, AZT) treatment for patients with AIDS and severe ARC provided the opportunity to study various features of infection with HIV-I 26. The placebo group was discontinued and offered zidovudine when it became clear that significantly more were surviving in the zidovudine-treated group. Jackson et al.*’ reported serial p24 antigen levels in 12 patients who had, or developed, antigenaemia ranging from 16 to 3006 pgml-‘. Zidovudine, 200 or 250 mg every 4 hours, reduced antigenaemia by approximately 90%; other dosage regimens were less effective. Although it was still possible to isolate virus from cultured lymphocytes during full dose zidovudine therapy this, and subsequent, studies** have shown a correlation between clinical benefit and an antiviral effect. de Wolf et a1.29 measured the effect of zidovudine on serum p24 antigen levels in 18 symptom-free men with longstanding HIV-l antigenaemia. The antigen level rose in only one patient and declined significantly in 13 (to below cut-off levels in nine). A virological effect, as judged by disappearance of circulating p24 antigen, was observed in patients with AIDS receiving foscarnet (phosphonoformate) by continuous infusion30, and some effect on antigenaemia was claimed in phase 1 studies of 2’ 3’dideoxycytidine 31. The lack of clinical response to suramin in patients with AIDS and ARC reported by Kaplan et aL3’ was supported by an absence of virological effect in a report from another group”. As none of these putative reverse transcriptase inhibitors can be expected to influence the existence of an integrated provirus in lymphocytes or other cells which are already infected, virus culture has little, if anything, to offer in their assessment in viva. As has been shown from studies conducted to date, if patients have pre-existing antigenaemia, monitoring of antigen levels during therapy may be used to assessactivity of the virus. It
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is likely, therefore, that as new drugs with acceptably low toxicity profiles are developed. their toxicity in humans and antiviral efficacy will be determined by phase 1 trials in small groups of HIV-l infected volunteers who are p24 antigen positive. Detection of infection in infants
There are problems in the diagnosis of HIV-l infection in the offspring of women who are seropositive. Persistence of maternal antibody, which may be detected for over one year by currently available ELISAs, may mask low levels of antibody production in the child, and there is no satisfactory specific IgM test as yet. In addition, virus isolation has been achieved from some infants who are totally negative by all antibody testing systems. Borkowsky et ~1.‘~ reported the finding of HIV- 1 antigenaemia in 9/58 children, with clinical, epidemiological or immunological evidence of infection, all of whom had negative antibody ELISA results. In a further report, these workers found p24 antigenaemia in 53% of asymptomatic children born to HIV-l antibody positive women, 70% of children with ARC and 85% of children who had experienced an opportunistic infection 35. It remains to be seen, however. whether antigen testing will be a useful addition to virus culture and sequential antibody measurement in assessment of infection in neonates and children. HIV-l
antigen in body fluids other than blood
HIV-l antigen can be detected in the cerebrospinal fluid (CSF) of some HIV-1 positive patients. Goudsmit et al.” reported positivity in all 5 children and S/9 adults with AIDSrelated encephalopathy, and negative antigen tests in 13 seropositive controls. However, longitudinal study of one of the controls showed that HIV-l antigen had been present transiently before the development of antibody in the CSF. These workers suggested that the persistence or reappearance of antigen in CSF may correlate with neurological deterioration, Others have confirmed the biphasic nature of CSF antigen positivity with an early transient rise followed by later persistence in some patients36. Rasheed et al. detected found p24 antigen in 5/7 (71%) of patients with encephalitis and 4/24 (16%) of patients without signs of central nervous system disease”; they pointed out, however, that the assay could not distinguish between encephalitis due to toxoplasmosis and that caused solely by HIV- I. It remains to be determined whether the presence and quantity of antigen in CSF can be used to assess the extent and prognosis of neurological damage by HIV-I and, possibly, intracranial opportunist infections. In the course of treating patients with AIDS and ARC with low-dose zidovudine (250 mg, 6-hourly), de Gans et ~1.~’ reported disappearance of HIV-1 antigen from CSF within 8 weeks in three patients who had previously been persistently positive. This occurred in the absence of significant neurological improvement. Interruption of therapy in one patient led to the reappearance of antigen which reached a high level and then disappeared when zidovudine was restarted. HIV- I antigen testing has been applied to other body fluids including saliva, bronchoalveolar lavage and semen in attempts to assess the level of virus excretion. Unfortunately the finding of a high degree of false positivity has hampered this work and it remains to be seen whether the test can be used for this purpose. Reports of positivity in broncho-alveolar lavage in lymphocytic interstitial pneumonitis” and not in other AIDS-related lung diseases require further investigation.
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D. J. Jeffriea Conclusions
The introduction of antigen tests for HIV-l infection has provided a valuable contribution to diagnosis of infection and an important predictive marker of disease progression. The occurrence of false-positivity, albeit at a low level in serum and plasma samples, necessitates the use of a neutralization or blocking test. The use of the test for determining prognosis must be handled with care, as some individuals can remain antigen positive and asymptomatic for years, but it may prove to be of value in selecting patients for trials and clinical use of new anti-retroviral agents as they become available. The presence of antigen in blood and CSF can be used as a marker for assessing the efficacy of anti-retroviral agents in vim, and, as far as can be judged to date, depression of antigenaemia has correlated with clinical improvement. As in other areas of AIDS research, technological advances are occurring rapidly and new tests, detecting antigens other than ~24, requiring smaller sample volumes, and having shorter incubation times and greater sensitivity levels, should soon be evaluated.
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