HIV-1 surveillance assays

HIV-l surveillance

assays

D. Aw Department

of Bacteriology,

University

Current Opinion in Immunology

Introduction With the spread of the acquired immunodeficiency syndrome (AIDS) worldwide, the importance of accurate laboratory methods for the detection of its main aetiological agent, the human immunodeficiency virus (HIV) type 1, was recognized. This led to the development of various immunoassays, initially measuring serum antibodies [the numerous enzyme immunoassays (EIAs) and western blot assays (W&As) are examples]. Although these assays are fast, cheap, and can be automated, other assays are needed for use in developing countries and on samples other than blood for large-scale epidemiological studies. In addition to the detection of the antibody response, alternative approaches, such as the detection of viral antigen or nucleic acids, have been developed recently to overcome some of the deficiencies of the antibcxiy tests, particularly in the early stages of infection.

Structure of HIV-1 HiV-1 belongs to the retrovirus family, which is charac-

terized by an RNA genome and a reverse transcriptase enzyme. The viral particles consist of antigenic glycoproteins anchored in a lipid. membrane, enclosing both an inner protein shell and other antigenic proteins (core proteins>. In addition, the particle contains two RNA molecules and the reverse transcriptase enzyme complex. After infection, a DNA transcript of the RNA molecule becomes incorporated into the host cell DNA. Once integration has occurred, there is no lmown method for eliminating the provirus. The virus infects T-helper lymphocytes and macrophages that carry the surface’CD4 antigen. Activation and release of virus expands the infection but also stimulates the host’s immune system. The result is that infected individuals make antibodies to a wide range of virus-encoded proteins.

of Edinburgh,

Edinburgh,

UK

1990, 233924397

developed for use in the mass screening of blood and blood products, are also used for individual diagnosis. When a serum sample is found to be consistently reactive by EL4 screening, confirmation from independent assays is needed. In many areas of the world, the WBA has been widely used for this purpose. In the initial diagnostic EIAs or ‘sandwich-type’ assays, HIV1 antigens were prepared from virus grown in lymphocyte culture: such preparations contained all the viral proteins. The most popular test format was to attach the viral extracts to a solid phase (plastic plate or bead); patient antibody bound to this and was detected with an anti-human serum to which enzymes (phosphatases, etc.) were often bound, although radioisotopes, fluorescein conjugates and biotin/streptavidin were also used. These were known as anti-globulin assays. There were two alternative formats for these assays: firstly, the competitive assay in which antibody in the patient’s sample competes with labelled anti-HlV antibody; and secondly, the antibody capture assays in which known immunoglobulins are captured by antibody onto a solid phase, and then allowed to react with labelled antihuman&G. False positives occurred in certain serum samples using both assay formats, e.g. in those from multi-transfused patients or patients with autoimmune disease, where the antibodies were directed against major histocompatibility complex class II antigens incorporated into the virus membrane. Parous women sometimes also shqwed false reactions, because of the presence of antibodies directed to the lymphocyte antigens stimulated during pregnancy. Non-specific reactions in these first-generation anti-globulin-type HIV-l assays were caused by the incorporation of, or contamination by, host antigen in the HIV-1 antigen preparations used in the coating of the solid phase; one example was the incorporation of the class II human leukocyte antigen (HLA) in the H9 cell line in which the virus is grown (Kuhnl et al, Luncet 1985, i&1222-1223). This led to the development of second-genetion

lmmunoassays antibodies appear 3-4 months after infection, which usually persists for life (Fig. 1). The EIAs, initially

Anti-HIV-1

immunoassays using monoclonal antibodies and cloned peptides. Recombinant virus protein replaced the lysate and, in one instance, the assay was a competitive EL4 [ 11.

Abbreviations AIDS-acquired

892

immunodeficiency syndrome; EIA-enzyme immunoassay; HIV-human immunodeficiency virus; HlA-human leukocyte antigen; IFA-indirect immunofluorescence assay; PCR-polymerase chain reaction; WBA-western blot assay.

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Current Biology ltd ISSN 0952-7915

HIV-1 surveillance assays Aw

893

Fig.1. Sequence of events in human im-

.

munodeftciency virus type 1 infection showing antibody profiles and virus/p24 levels from infection to the onset of the acquired immunodeficiency syndrome (AIDS).

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In this assay, there was competition for binding to the immobilized recombinant antigen between the anti-HIV1 antibodies in the sample or control and the enzymelabelled human monoclonal antibody. Cellular contam ination was excluded by the use of a recombinant envelope protein (gplbo), at the same time enhancing the target epitope at the solid phase. In this assay, the human monoclonal antibody (CBHIV-lgp41) recognized an immunodominant epitope on the transmembrane glycosylated protein gp41 (which also induces an immune response in HIV-infected people) and demonstrated crossreactivity with gp120, the major envelope glycoprotein, it could therefore compete for two binding sites on bound recombinant gp160 with antibodies directed against gp41 and gp120. Unlike the first-generation assays, most of which preferentially detect IgG after seroconversion (4-6 weeks after infection), the competition type assay allows the detection of an IgM response, this being the first-stage immune response after HIV infection (Fig. 1). The Wellcozyme EL4 was the lirst assay to be both first-generation and competitive. There is evidence that the antibodies in the early stages of infection include antibodies of the IgM class and it may be relevant to be able to detect these in any assay for detecting HIV-l. The development of an anti-HIV-1 IgM detection system has been described in this system, by Healey et al (AZDS 1989,3:301-304); a mod&d particle agglutination assay detects anti-HIV-l IgM up to 10 days earlier than some EIAs.

The western blot assay although basically another form of the antiglobulin assay, is the most widely used confirmatory assay because it produces a profile of antibody reactivity to

The WI34

speciIic antigens during HIV-l infection without the need to handle infectious viral particles or radioisotopes. Comparable sensitivities have been reported for the WBA and the indirect immunofluorescence assay (IFA) when used as conIirrnatory assays [ 21. In another comparison [3], the sensitivity of the WBA and the IFA appeared to improve after modification of the procedure used to harvest cells from blood samples. On the basis of the assumption that viral antigen appearance on lymphocytic or monocytic membranes occurs in the early stages of infection, mopping up low titres of specific antibodies, blood was collected from at-risk patients who were both antibody-negative and antigen-negative. Cells were separated by centrifugation, then washed in Hank’s solution and eluted in acid medium. In two of the eighteen subjects, bands suggestive of HIV infection appeared in the WBA when the eluates were run. It was suggested that this test might be useful in the detection of the earliest stages of the immune response to HIV. However, there have been cases of uninterpretable or atypical western blots, in which the provirus has been shown to be functionally defective [4]. The WBA has several other disadvantages. These include the fact that it is not possible to quantify the results and the subjectivity.involved in reading the assay. WBAs are also labour-intensive. In addition, there is the selective disappearance in vitro of gp120 from virus particles used as an antigen source (giving rise to false-negatives), as well as the presence of contaminating human cellular antigens. The loss of gp120 from virus particles is important diagnostically as antibodies to the envelope proteins &pI2O/gpI60) play a major part in the humoral response to infection. Difficulties are also encountered in the transfer of large molecules to nitrocellulose (Poxton, this issue, pp 905-909).

,

Potential confwmatory assays The above problems have been resolved in a variety of

Alternative approaches

ways, Including the use of cloned peptides and the binding of more than one viral protein to the solid phase to detect variant strains.

sitivity, they can be quite expensive and some, such as the EIA, require expensive equipment to optimize sensitivity. The World Health Organization has requested the development of less expensive assays, especially for countries that cannot afford routine screening for HIV-l by EL4 or WBA as currently available, although certain particle agglutination tests are cheaper to use.

Antibody responses to HIV-l antigens vary, depending on the individual and the course of disease progression. Therefore, even in assays in which a single synthetic peptide represents one immunodominant epitope or a single recombinant antigen represents several epitopes, there may be a failure to detect antibodies in people infected with variant strains of HIV-l, or to detect antibodies to other epitopes. One way round this problem has been to use J?sc&&& c&expressed fusion proteins from subcloned proteincoding regions representing the three main genes: gag, poJ and env [5]. In this study, six antigens were expressed as fusion proteins with the first 56 residues of E. coli galactokinase. Microtitre wells were coated with the six purified antigens. The N-terminal galactokinase peptide (gaK-56) and E. cofi crude extract were added to each sample to neutralize antibodies that cross-reacted with galactokinase or other co-purified E. coli antigens (in the absence of Gaul-56, iive out of 1016 seronegative patient samples were positive). No false-negatives were reported, and there were fewer indeterminate results from 143 samples from patients with AIDS and AIDSrelated complex than produced by the Dupont WBA No false-positive results were observed in the 1016 specimens from seronegative donors.

Although all the above tests show high specificity and sen-

There are several possibilities for cheaper irnmunoassays. One, an immunodot blot assay, only requires microlitre quantities of serum. Antibodies are detected by nanogram quantities of HIV lysate immobilized on nitrocellulose paper [7]. This is helped by the strong hydrophobic association between protein and nitrocellulose. Colour develops from the action of horseradish peroxidase-conjugated goat anti-human IgG and chloro1-naphthol with hydrogen peroxide. The limit of detection for positive sera ranges from 15 to 30ng of antigen on the filter. The assay gave comparable results to a commercial EIA (Bio-Enzabead) and a non-commercial EIA, with all three tests detecting serum antibody up to a dilution of 1 in 106. In 50 samples (33 antibody-positive and 17 antibody-negative), it produced results of identical sensitivity to the WBA There are three main advantages of this system it is economical compared with commercial EIAs; it only requires small amounts (l-2 pl) of sera; and expensive equipment is not required to analyse the results. Other possibilities include the use of saliva in an IgG antibody-capture radioimmunoassay (Johnson et al, AIDS 1988,2:369-371) and the detection of anti-HIV-specific IgM by a mod&d particle agglutination assay in which IgM is detected 1-2 weeks after acute symptoms appear (Healey et al, 1989).

In another EL4 [6], the complete glycosylated gp120 and/or gp160 molecule was used as the capture antigen. These molecules exhibited the maximum number of epitopes available for binding by antibodies. In addition, they bound to the solid phase in conformations that were more likely to be found in the native protein than is the case for Individual or synthetically produced peptides. Different strains of HIV-l were used as sources of env antigens. In one serum sample, the gpl2Os of HTLV-IIIB, NYS/LAV-1, HTLV-IIIRF, SF-33, SF-2,284,2129, CBL4 and HTLV-IIIMN but not of the HIV-2 strain CBL20 were detected. The EIA allowed the detection of all these strains although the percentage amino-acid divergence from the HTLV-IIIB (~~10) sequence in the envelope region is 3.2% for LAV-1 (BRU), 15.2% for SF-2, 15.4% for HTLV-IIIMN and 17.7% for HTLVIIIRF (divergence calculated from Los Alamos data bank by counting unmatched amino acids as dissimilarities). The percentage amino-acid divergences of the African strains 284, 2129 and CBL4 are thought to be greater in this region (although the sequences have yet to be reported) but these strains were still detected by the assay.

In many cases where there are low particle numbers early in infection, there may be a reduced or non-detectable Immune response. With HIV-l, the viral DNA is integraud into the cellular genome and there are very few other laboratory markers of viral presence. In these cases, the ability to detect small amounts of virus is useful. - ”

A possible development of this assay might be as a monitoring system for the early stages of seroconversion, when env antibodies appear before or along with those to gag proteins. The assay can also be used to monitor the humoral immune response to candidate HIV-l vaccines which incorporate gp120 and/or gp160 derived from divergent HIV-1 strains.

The PCR technique was invented by Kary Mullis of the Cetus Corporation (Saiki et al, Science 1985, 230:135&1354; Mullis and Faloona, Meth EnzymoZ 1987, 155:335-350). The reaction is an in vitro technique of nucleic acid amplification speciiically applied to a particular segment of DNA Two synthetic oligonucleotide primers that will hybridize to the ends of opposite strands of the

The polymerase

chain

reaction

There has been great interest in methods for the direct detection of nucleic acids. The polymerase chain reaction (PCR) is one such method and is useful for viruses that are diihcult or impossible to culture or for those with limited antigen production. The major requirement is that at least part of the sequence of the nucleic acid is known.

HIV-1 surveillance assavs Aw

target sequence are required. As these are situated so that extension by the polyrnemse occurs between them, the size of the amplified product is determined by the distance between the two primers. The process is cyclical, involving three stages (Fig. 2). The double-stmnded target DNA is iirst denatured at 9095°C (step l), then cooled to 37-50°C. At this temperature, the primers anneal to the target DNA strands (step 2). The DNA strands can now be copied and the primers extended with the thermostable Taq polymerase (purified from the thermophilic bacterium, -us uquutk~~) at 67-72°C (step 3). The value of this enzyme is that it is relatively unaffected by the denaturation step and therefore does not need to be replaced at the end of each cycle. The products at the end of each cycle will contain the complementary stmnds for each primer and the reaction therefore consists of cycles of denaturation, annealing and extension. The ampliiication of the DNA is exponential, allowing up to a million-fold or more increase in 25-35 complete cycles. This increase can be calculated as approximately 2’: where 92is the number of cycles of amplification performed (Fig. 3). One important use of the PCR has been in the early detection of HIV-l proviral sequences in infants born to seropositive mothers. This is important when early treatment of the virus is likely to be of benefit. Of the infants born to these women, l&50% will acquire the virus in utero, during labour and delivery, or post-partum through breast-feeding. The diagnosis of HIV infection is diificult because of the passive acquisition of ma ternal antibodies, which persist for up to 15-18 months (Scott et al, JAMA 1985,253:363366; The European Collaborative Study, Luncet 1988, ii:10391043). Also, a child may be infected even if antibody disappears. It is therefore important to use methods that detect virus directly rather than rely on the antibody response. Antigen assays

and virus culture may have a role but are not universally applicable or available. One advantage of the PCR is that only a small amount of sample is required [ 81; this proved useful in the case of neonates (mean 5.7-16 days of age in this study), from whom only small volumes of blood (0.5-2 ml of peripheral blood mononuclear cells) can be collected. In this study [8], blood was also collected during the iirst 2 years of life ln some infants with seropositive mothers. The DNA from 0.2 ml of blood was used for each reaction and the primer pair (X38/.X39) was specilic for the gag p24 sequence. The study covered the neonatal and postnatal periods. PCR was used to detect proviral sequences in the infants before progression to AIDS or, in certain cases, development of non-specific signals (failure to thrive, persistent oral candidiasis, etc.) suggestive of HIV infection. The proviral sequences were detected In live out of seven neonates and in six out of six infants in the postnatal pe nod (median age, 5 months) in whom AIDS later developed. PCR also detected one out of eight neonates and four out of fourteen infants in the postnatal period who developed the non-specific symptoms of HIV infection. In 25 infants who remained well after a mean follow-up period of 17 months, no HIV proviral sequences were detected. Therefore, the results of this study [8] show that PCR could be used as a diagnostic test to indicate that early treatment is required, e.g. with HIV hyperimmune globulin or prophylactic antiviral therapy, where treatment may be more effective if given soon after infection (Koff and Hoff, Science 1988,241:426-432). The test could also be used as a means of predicting the progression to AIDS in these infants, PCR and nucleic acid detection by in situ methods have also been shown to be useful in seronegative patients at Fig.2. Human immunodeficiency virus (HIV)-1 DNA amplification by the polymerase chain reaction. Step 1, DNA duplex denaturation; step 2, primer annealing; step 3, DNA extension by Taq polymerase.

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Immunological techniques

p

Unamplified DNA Targeted sequence

c

Cycle 1 -

Segment 1 and 2 Denature and Segment 3

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anneal primers Primer extension

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Although the PCR is sensitive in amplifying specific sequences, one major problem is the inadvertent contamination of samples by positive target DNA (either known or unrecogniz~ positives alongside negatives), probably during sample preparation. This produces false-positives and occurs pnmarily with the use of air displacement pipettors and carryover from glove lingertips. PCR can amplify DNA by between a million and a billion-fold and aerosols are produced when reaction tubes are opened, so that PCR products accumulate in the laboratory. There are certain guidelines that should be followed to minimize contamination of reactions [ 101. These include the separation of pre-isolation and post-isolation work areas; the use of positive displacement pipettors; aliquoting all reagents; using well characterized controls; checking the reproducibility of results; and using nested primers.

Cycle 3 1

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by EIA, WBA and antigen capture), four tested positive with both the DNA-DNA and the RNA-RNA techniques as well as by PCR (using the SK68/SK69 primer pair). Samples from selected blood donors were negative. These results show that both in situ hybridization techniques and the PCR can be useful additional tools in the diagnosis of high-risk asymptomatic seronegative carriers.

Denature and _

--

anneal primers

Primer extension

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‘Short product’

p Cycles 4-25

‘Long product’

4 At least 105-fold increase in DNA

Fig. 3. Amplification of target DNA by the polymerase chain reaction.

risk of infection where, for example, sexual partners are infected with HIV but are negative by current serological assays, i.e. ‘silent’ HIV infection (Soriano et al, Luncet 1990, i&O).

high

In another study [91, Iive patients were tested: two female sexual partners of HIV-infected men; a bisexual man who had sexual intercourse with infected partners; and two infants (18 and 24 months old) of asymptomatic seropositive mothers. Two in situ hybridization techniques for detecting DNA and RNA in blood lymphocytes were compared with the PCR A pSP64 plasmid BHlO recombinant clone was used as probe. Of the Iive patients (all negative

Several suggestions have been made about the use of DNA in the PCR One study [ 111 suggests the mod&ation of the positive control primer-annealing site so that, in the event of carryover, the control DNA is not amp&d with the normal primer pair. Other suggestions include the use of another pair of primers, one of which is complementary to the vector DNA A positive result with these vectors indicates contamination [ 101. Contamination aside, the uses to which the PCR has been put are steadily increasing in number. RNA can now be used as the starting material for PCR amplilications (Ferre and Garduno, Nucleic Acid Res 1989, 17:2141). PCR has also been used in HIA typing with allele-speciiic oligonucleotide probes on PCR-ampliiied DNA HLA class II genes can now be routinely analysed using published sequences and sequence-specific primers [12]. It has also been used to quantify the number of HIV provfral sequences in circulating mononuclear cells; this had only been guessed at previously (Simmonds et al, J Viroll990,64:864-872). A recent book by Innis et al, (PCR Protmols.~A Guide to Methods and Appliuations Academic Press, 1990) provides an excellent guide to all aspects of PCR and suggests future applications.

Conclusions ,* It can be seen that progress in the field of serological HIV stuveillance in the last year or so has involved the use of cloned or synthetic peptides, monoclonal antibodies, and possible developments in dot-blot protocols. PCR has yet to be used diagnostically but has shown itself to be a useful tool in the research field.

HIV-1 surveillance assays Aw

Annotated

references

and recommended

reading

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Of interest Of outstanding interest

1.

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897

envelope glycoproteins of divergent strains of HIV-l. AIDS 1989, 33155-163. An EL4speckic for gp12O/gp160 was developed using gp120 as the capture antibody. Sensitivity of the assay reached dilutions of 1300000 in HIV-positive sera. XU J-Y, GORNYMK, EOnA-PAZNER S: An immunodot blot assay for the detectIon of antibndy to HIV. J Immunol Methods 1989, 120:179183. A possible alternative assay to EIA, this is an Immunodot blot assay which o&y requires nano8ram quantities of capture antigen and microiitre quantities of patient serum to detect HIV-1 antibodies. 7.

S-H, PO~TMANNT, GRUNOWR, JUNGBAUER A, VON ?? BAEHRR: AppIication of a human monocIonaI antibody in a rapid competitive anti-HIV RLISA J Immwwl Me&c& 1989, 116:229-233. Reports the development of an akernatiwz, fast competitive EL4 using both recombinant HIV envelope protein and a monoclonal antibody capable of detectir18 IgM and I@%. 2. 0

N DS: Evaluation of two YANKEY RJ, KEISER JF, KIMJ-J, W~XINSO confirmatory assays, Western blot and indirect fluorescence for the detection of human immunodeficiency virus (HIV1) antibodies. Diqn C&n Test 1989, 12748-50. When used on 735 sera, the WBA and IFAwere comparable in sensitivity and speckicky. BEDARIDAG,

PANZ.ERI MP, Irzton A, D’~TLNo F, CAMBIEG, NARDEUAML, BERTOE, CROCCH~OLO P: Detection of HIV antibodies in seronegative subjects at risk for HIV infection. AIDS 1989, 3613-617. Describes methods for detecting low levels of HIV antibodIes by WBA In EIAseronegative patients.

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HUET T, Dw M-C, BRUN-VEZJNFZ F, ROEIANISGE, WAIN?? HOBSONS: A hi@y defective HIV-1 strain isolated horn a healthy Gabonese individual presenting an atypical Western blot. AIDS 1989, 3707-715. Reports the isolation of an unusuai HIV-1 strain from a healthy Gabonese present@ with an atypical WBA The IsoIate was apparently non-cytopathic and did not seem to stimuIam em-speciiic antibodies.

CHMNGCS, GROVET, COOPERM, CUANJ, Kow~L~taA, PARCELLS ma K, TSUNOKAWA M, ROSENBERG M, Aacutu E, FRANI(uN S, Shcrn T, DEBROUCK C: Development of a con6rmatory enzyme-Iinked immunosorbent assay for HIV-l antibodies. Clin Qk?m 1989, 35946-952. Six protein-cod@ regions from the gag, env and pd regions of HIV-1 were expressed as fusion proteins in E c&with the first 56 residues of @actokinase and then used as coating antigens for a confirmatory EIA. 5.

6. .e

MOOREJP, WN.LQZELA, FO~IEIT EAC, MCKF.ATING J: An enzyme-Iinked immunosorbent assay for antibodies to the

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ROGERS MP, Ou C-Y, RAYFEI.D M, THOM& PA, SCHOENBAUM EE, ABM E, KRASINSKI K, SELWYN PA, MOOREJ, KAUL

G. THE NEW YORK A, G~UMMKT, BAMJIM, ficHOCHETMAN ClTY COLIABoRATIvE STUDYOF MAmRNALHIV TRANsMlsslON ANDMONIE~OREh&DIcAL CENITCEHIV PERlN4TAL?-RANShtISSION STUDY GROUP: Use of the poIymerase chain reaction for early detection of the provimI sequences of human immunode&ciency virus in inhmts born to sempositive mothers. N En@ J Med 1989, 320:164%1654. PCR was uxd in the detection of HIV proviraI sequences in infants from seropositive mothers dur@ neonatal and postnatal periods. This method overcame the problem of detectIn8 maternal antibodies and may possibIy provide an indicator of progression to AIDS. PEZZEL~AM, R0s.w P, LOMBARDI V, GEMEUJ V, I+&&~NI COSTANTINI R, MIROLO M, FUNDARO C, MOSCHESE V, WIGZEUH: HIV vimI sequences in seronegative people at risk detected by L situ hybridisation and polymerax chain reaction. Br Med J 1989, 298:713716. The use of in situ hybridization and PCR to assess latent infection in hi&risk serone@ive patients.

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Cmwmv JP: The poIymerase chain reaction a rwiew of the practkzd Iimitations for human Immunodeficiency virus diagnosis. J Wol Met&oh 1989, 25:179-188. Background and applications of the PCR 10. ??

OsTs C: ControIIing false positives in PCR. Dtiagn Clin Test 1989, 2750. A short paper contaInin suggestions about how to detect fake-positive si8nals in PCR 11.

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BELLJ: The poIymerase chain reaction. Immund 12. 0 10:351-355. Immunolo@aI applications of PCR

To&y 1989,