Lepr Rev (2007) 78, 137–147

Serum samples from patients with mycobacterial infections cross-react with HIV structural , p55 and p18 TAHZIBA HUSSAIN, SHIKHA SINHA, KIRAN KATOCH, V. S. YADAV, K. K. KULSHRESHTHA, ITU SINGH, U. SENGUPTA & V. M. KATOCH HIV/AIDS UNIT & Clinical Division, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (Indian Council of Medical Research), Tajganj, Agra 282001, India

Accepted for publication 31 October 2006

Summary Background Infection with Mycobacterium leprae is associated with a high frequency of false positive results in a variety of serological assays. Our studies have found cross-reactivity to HIV structural proteins in serum samples from leprosy patients, irrespective of the type of disease, treatment duration, age and gender and from a few patients with active TB disease. Methods Western blot (WB) analysis revealed that sera from HIV negative leprosy patients across the spectrum showed high reactivity with p18, Gp41 and p55 and lower reactivity with other HIV proteins. The reactivity appeared to be specific; western blot-positive samples were negative in ELISA and in several rapid tests for HIV. Cross-reactivity was not found in sera from patients with leishmaniasis or from normal healthy individuals. Results None of the WB reactive leprosy patients seroconverted to HIV positivity within 6 months to 1 year after Western blot testing. BLAST analysis revealed that envelope antigens of HIV (Gp41, Gp120 and Gp160) contained amino acid sequences similar to M. leprae ML0470, putative integral membrane , Rv0740, mmpL9 (M. tuberculosis). Core (gag) antigens (p18) had similarities to ML0406, but polymerase antigens (p52) had similarities to PE_PGRS (M. tuberculosis, H37Rv). Nucleotide sequence analysis, on the other hand, did not reveal any significant homology between M. leprae or M. tuberculosis and HIV. Conclusions The occurrence of these high false-positive rates in M. leprae-infected individuals suggests a possible complication of serodiagnosis of HIV in regions where mycobacterial infections are endemic. There is a need for caution in reporting HIV infection among leprosy patients. Our observations emphasise the value of the various rapid assay kits for HIV, where this false positivity is not observed.

Correspondence to: T. Hussain (Tel: þ91 562 2331751-4, ext. 287; Fax: þ91 562 2331755; e-mail: [email protected])

0305-7518//064053+11 $1.00 q Lepra 137 138 T. Hussain et al. Introduction

Several authors have studied the potential effects of HIV infection on leprosy infections. Studies on the interactions of these two diseases assume importance in regions where both diseases are endemic. Surveillance studies at our Institute showed that the incidence of HIV infection among leprosy patients remains low, although there has been a slight rise in HIV positivity from 0.12% (5/4025) during 1989–1993 to 0·38% (8/2125) during 1999–2004. Follow-up of these patients after 6 months showed that downgrading in the clinical spectrum to a more severe form of leprosy did not occur. Neither reversal and ENL reactions nor neuritis (chronic or acute) was observed among the HIV-positive leprosy patients, nor did any develop ARC or AIDS.1,2 The present study is an extension of an earlier finding by Shivraj et al.3 that antibodies to HIV-1 specific p24 antigen were present in sera from leprosy patients (14/43), TB patients (1/21) and contacts of leprosy patients (2/25). No reaction was observed to Gp120, Gp160 and p31 antigens. In order to explore this systematically, we have now studied 100 serum samples from leprosy patients, across the spectrum of disease, as well as from patients with tuberculosis (TB), leishmaniasis (post-Kala-azar dermal leishmaniasis, PKDL) and from normal healthy controls. We investigated the possible serological reactivity of these samples with HIV structural proteins.

Material and methods

Blood samples were drawn aseptically by antecubital venipuncture from leprosy patients attending the Unit-1 of Outpatient’s Department (OPD) of the National JALMA Institute for Leprosy & other Mycobacterial Diseases, Agra, after obtaining informed consent. The patients were classified as tuberculoid (TT), borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous (BL), lepromatous (LL) and neuritic (N) leprosy according to Ridley–Jopling criteria.4 The leprosy cases in the study were under surveillance after being treated with a full course of standard anti-leprosy multi-drug therapy, for periods of between 12–24 months. Samples were also collected from 10 patients with active TB disease attending the OPD of TB Demonstration, Research & Training Centre (TBDTC), Agra and from 10 patients with leishmaniasis. The inclusion criterion was for adult patients aged between 16 and 48 years. Children aged less than 15 years, as well as weak and very old persons were excluded, as being less likely to be sexually active. Socio-demographic status and clinical presentation at the time of sampling were recorded. Control blood samples were from 10 adults, (6 males and 4 females) aged between 20 and 50 years, HIV-negative by ELISA as they had no risk factors for HIV infection and with no history of mycobacterial infection. The blood samples were centrifuged at 2500 g for 10 min and the sera obtained were stored at 2208C until the assays were performed. The serum samples were screened for HIV-1/2 antibodies by ELISA. Those found negative were analysed by western blot (WB) and by rapid assays for HIV. All the WB reactive leprosy patients were re-examined after an interval of 6 months to 1 year using ELISA and rapid assay kits to assess HIV status. ELISA testing used a Clone Systems kit (Bio-Chem Immuno Systems, Italy). Rapid assay kits used were: HIV Capillus latex aggregation assay, Unigold (Trinity Biotech, Ireland), and Tridot (J. Mitra & Co. Ltd, India). Western blot assay kits were BIO-RAD (NEWLAVBLOT, Cross-reaction with HIV structural proteins 139 Tokyo, Japan) and QualiCode HIV-1/2 western blot kit (Transasia Bio-Medicals Ltd., Mumbai, India). Tests were carried out strictly according to the kit specifications and manufacturer’s instructions.

STATISTICAL ANALYSIS

Pearson’s chi-squared test was applied to determine the statistical significance of WB reactivity to HIV structural proteins among the different types of leprosy patient. The significance of differences in cross-reactivity among different categories was also examined with Z-statistics at 5% level of significance.

Results

Serum samples from 100 leprosy patients across the spectrum, HIV-negative in ELISA, were tested by western blot using the BioRad kit. The samples reacted to a wide range of HIV proteins, producing one, two or multiple bands (Table 1). To check the specificity of this finding 15 serum samples, three each of BT, BL, BB, LL, and neuritic patients, were tested using a second western blotting kit (HIV-1/2 Transasia). The sera reacted with a few proteins, viz., p18, p55, Gp41, Gp120 and Gp160. WB was also carried out on sera from 10 young leprosy patients, aged 9–15 years. Reactivity to p18, p24, p55, Gp41, Gp120 and Gp160 was observed even in these serum samples, showing that the phenomenon was not age-specific. Analysis of WB reactivity of leprosy patients’ sera to the various envelope, endonuclease and core antigens of HIV (Table 1) showed that 80% of all patients reacted with p18, 76% with Gp41 and 67% with p55. Forty-seven percent reacted to p52, 40% to p25, 36% to p68

Table 1. Western blot reactivity of sera from leprosy patients with HIV structural proteins

Type of Leprosy patients and WB Reactivity (%) (n ¼ 100)

HIV structural BT BL BB LL N Statistical analysis Proteins (n ¼ 20) (n ¼ 20) (n ¼ 20) (n ¼ 20) (n ¼ 20) (P value)

Env Gp 41 16 (80) 17 (85) 18 (90) 13 (65) 12 (60) P , 0·0001 Gp120 3 (15) 0 (00) 4 (20) 0 (00) 0 (00) Gp160 2 (10) 3 (15) 4 (20) 1 (5) 1 (10) Gag (core) p18 18 (90) 18 (90) 18 (90) 12 (60) 14 (70) P , 0·0001 p25 10 (50) 6 (30) 10 (50) 8 (40) 6 (30) p40 3 (15) 2 (10) 6 (30) 3 (15) 0 (00) p55 15 (75) 14 (60) 18 (90) 10 (50) 12 (60) Pol p34 6 (30) 5 (25) 6 (30) 3 (15) 2 (10) P , 0·004 p52 12 (60) 14 (70) 16 (80) 3 (15) 2 (10) p68 8 (84) 9 (40) 14 (70) 4 (20) 2 (10)

Env-Envelope antigens: Gp41, Gp120, Gp160 Pol-Polymerase antigens: p34, p52, p68 Gag (core)-Group associated antigens: p18, p25, p40, p55 Positive: 2 Env ^ Gag ^ Pol; Indeterminate: 1 Env ^ Gag ^ Pol, Gag ^ Pol, Gag, Pol; Negative: Non-Classified bands, No bands. 140 T. Hussain et al. and 22% to p34. A smaller proportion of the sera also reacted with p40 and env proteins, Gp160 and Gp120 (14%, 12% and 7%, respectively). This suggests that the core antigens, p18 and p55 and the env antigen, Gp41 of HIV-1 might share epitopes with M. leprae proteins recognized by all types of leprosy patient. WB reactivity of sera from tuberculosis patients to antigens of HIV (Table 2) revealed that the reactivity to env, gag and pol occurred in some patients with active tuberculosis but with no HIV infection. Bands for p24/p25, p34, Gp41, and Gp160 and Gp120 were observed. No reactivity was detected in the sera from 10 normal healthy controls, or in sera from 10 patients with leishmaniasis. None of the WB-reactive serum samples from leprosy, tuberculosis or leishmaniasis patients or from normal healthy controls showed reactivity in any of three rapid assays: HIV Capillus latex aggregation assay, Unigold and Tridot. This absence of misleading cross- reactivity emphasises the value of such rapid assay kits, which are available in this region. Follow-up of the leprosy patients included in these studies, after an interval of 6 months– 1 year, was done using ELISA kits and rapid assays. The follow-up period depended on the patient’s OPD attendance, and varied for several reasons but none of them was lost to follow- up. None of the WB-reactive leprosy patients seroconverted to HIV positivity. BLAST analysis (Appendix 1a) indicated that the envelope group of antigens of HIV (Gp41, Gp120 and Gp160) had aminoacid sequences similar to those found M. leprae ML0470, putative integral membrane protein Rv0740 and mmpL9 of M. tuberculosis. Core (gag) antigens (p18) had similarities to ML0406 and the polymerase group of antigens (p52) had similarities to PE_PGRS of M. tuberculosis (Table 3). Nucleotide sequence analysis, on the other hand, showed no significant homologies or similarities between M. leprae and HIV or between M. tuberculosis and HIV.

Table 2. Western blot reactivity of sera from patients with Tuberculosis, Leishmaniasis and Normal Healthy Controls with HIV structural proteins

WB Reactivity (%) of patients with Tuberculosis, Leishmaniasis and Normal Healthy Controls

Tuberculosis Leishmaniasis NHC Statistical analysis HIV structural proteins (n ¼ 20) (n ¼ 10) (n ¼ 10) (P value)

Env Gp 41 4 (20) 0 0 P , 0·042 Gp120 4 (20) 0 0 Gp160 4 (20) 0 0 Gag (core) p18 2 (10) 0 0 P , 0·05 p25 4 (20) 0 0 p40 0 0 0 p55 0 0 0 Pol p34 2 (10) 0 0 P , 0·04 p52 0 0 0 p68 0 0 0

Fig. in parentheses represents % reactivity. Env-Envelope antigens: Gp41, Gp120, Gp160 Pol-Polymerase antigens: p34, p52, p68 Gag (core)-Group associated antigens: p18, p25, p40, p55 Positive: 2 Env ^ Gag ^ Pol; Indeterminate: 1 Env ^ Gag ^ Pol, Gag ^ Pol, Gag, Pol; Negative: Non-Classified bands, No bands. Table 3. BLAST Analysis: Aminoacid Sequence Resemblances between HIV and M. leprae TN and M. tuberculosis H37Rv by BLASTP

Sequence match Length Score Bits Expect value % Identities % Positive Gaps

Env Gp 160, Gp 120, Gp 41 M. leprae ML0470 96 27·7 (60) 0·003 23/75 (30%) 39/75 (52%) 8/75 (10%) Putative integral membrane 983 26·6 (57) 0·006 15/51 (29%) 26/51 (50%) 6/51 (11%) proteins structural HIV with Cross-reaction protein Rv0740 175 26·6 (57) 0·006 8/35 (22%) 18/35 (51%) – mmpL9 (M. tuberculosis) 962 26·2 (56) 0·007 31/100 (31%) 48/100 (48%) 12/100 (12%) Pol p34, p52, p68 PE_PGRS (M. tuberculosis) 923 33·9 (76) 0·006 27/89 (30%) 41/89 (46%) 10/89 (11%) Core (Gag) P18, P25, P40, P55 ML0406 224 53 (23·7) 0·083 16/50 (32%) 22/50 (44%) – ML0406 106 54 (24·1) 0·053 16/50 (32%) 23/50 (46%) – 141 142 T. Hussain et al. Discussion

HIV infection constitutes a major risk factor for tuberculosis and for some other mycobacterial infections such as Mycobacterium avium-intracellulare, and an association between the viral and mycobacterial infections has been established.5,6 However, there are still uncertainties regarding the interaction of HIV with leprosy.7,8 HIV infection is known to lower the CD4 þ T- lymphocyte count resulting in a generalised immunosuppression, especially of T-cell mediated immunity.9 This lowers resistance to a wide range of opportunistic and other infections. Therefore, it is possible that HIV infection might play a major role in lowering the immune response in leprosy patients and expansion of the HIV epidemic into leprosy-endemic areas could have a significant effect on the epidemiology of leprosy. Diagnosis of HIV infection using WB assays is subject to controversy.10 –12 The technique is not routinely used as a screening tool because it yields an unacceptably high percentage of indeterminate results. Indeterminate or atypical patterns are characterised by reactivity with group-specific (core) viral proteins antigens. WB may be used as a confirmatory test for HIV infection but lacks standardisation, is cumbersome, and is subjective in interpretation of banding patterns.13 – 15 The serological diagnosis of infection is usually made on the basis of the detection of circulating antibodies specific for viral antigens gp41, gp120 and gp160, but despite the introduction of recombinant immunogenic oligopeptides, which improve the sensitivity and specificity of immunological tests, both false positive and false negative reactions have been reported.16 Andrade and colleagues have reported a pilot study of HIV-1 serology in 57 multi-bacillary leprosy patients (LL and BB) and found that 4/57 (7·02%) were reactive on first testing but were non-reactive on second testing.17 Many studies have evaluated the validity of test results using either ELISA, rapid tests or WB18 – 23 and have reported cross-reactivity between HIV antigens and other disease conditions such as lymphoma, leprosy, biliary cirrhosis, systemic lupus erythematosus, viral hepatitis, multiple sclerosis, influenza, autoimmune diseases, renal failure, blood transfusions, transplantations, injection drug use, alcohol-related liver diseases, multiple pregnancy, malignancies, and malaria.24 –32 Reactivity to human placenta has also been found33 and cross-reactivity between HIV and sera from non-human animals including cattle, goats, alloimmune mice and dogs has been noted.34 –36 The present study indicates that a significant proportion of leprosy sera, without HIV infection, react with several HIV-1 antigens. Some sera from patients with active TB disease also react, but this is less frequent. Therefore, we feel that there is a need for caution in reporting HIV infection among leprosy patients where evidence for infection is based on WB. However, we do not claim that leprosy patients give false positivity in test for HIV. Our studies have found cross-reactivity, detected by WB, in sera samples from leprosy patients that were negative by ELISA, irrespective of the type of disease, treatment duration, age and gender. BLAST analysis revealed that the envelope group of antigens of HIV (Gp41, Gp120 and Gp160) had aminoacid sequences similar to proteins found in M. leprae and M. tuberculosis, as did Core (gag) antigens (p18). Some of the observed similarities appear significant. Nucleotide sequence analysis, on the other hand, did not reveal any significant homology or similarity between M. leprae and HIV or between M. tuberculosis and HIV. The resemblances between the proteins might explain the results we have obtained by WB, and could be functionally important in molecular mimicry or receptor binding. Further study of Cross-reaction with HIV structural proteins 143 the significance of these resemblances, and of whether they explain some of the ‘false positives’ obtained by WB, seems warranted.

Acknowledgements

This study was supported by funds from the Indian Council of Medical Research, New Delhi. Shikha Sinha is a recipient of the Senior Research Fellowship of the Council of Scientific and Industrial Research (CSIR). The authors thank Mr. K.L. Verma, Mr. M.M. Alam, Mr. P.N. Sharma and Mr. M.S. Tomar of the HIV/AIDS Unit and the entire staff of OPD for their assistance in the study.

References

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Appendix 1. Blast analysis

A comparative proteomics of M. leprae, M. tuberculosis and HIV, by a detailed basic local alignment search tool for proteins (BLASTP) of the leprosy and tuberculosis and HIV, might give an insight into the common regions of the three which, in turn, would be helpful in developing a diagnostic tool or a vaccine for one and/or all of these highly infectious diseases. Therefore, BLAST analysis and nucleotide sequence analysis was carried out. Potential coding sequences were predicted and and protein sequences analysed as prescribed previously, using Artemis to collaborate data and facilitate annotation. The genome and proteome sequences of M. leprae and M. tuberculosis H37Rv were compared pairwise to identify conserved genes using the Artemis Comparison Tool. The sequence was assembled using Phrap (P. Green, published), finished using GAP4, and compared with sequences present in public databases using FASTA, BLASTN and BLASTX. EMBL databank accession number for the sequence is A L450380. Cross-reaction with HIV structural proteins 145 pol Score E Sequences producing significant alignments: (bits) Value gij15608117jrefjNP_215492.1j PE_PGRS [Mycobacterium tuberculosis 34 0·006 gij15608117jrefjNP_215492.1j PE_PGRS [Mycobacterium tuberculosis H37Rv] Length ¼ 923 Score ¼ 33·9 bits (76), Expect ¼ 0·006 Identities ¼ 27/89 (30%), Positives ¼ 41/89 (46%), Gaps ¼ 10/89 (11%) Query:2 QITLWQRPIVTIKI-GGQLKEALLDTGADDTVLEEMNLPGKWRPKMIGG IGGFIKVRQYD 60 Q þ P þ VIþ GGQ þ LLDTG þ V þþL þ P þ GG þ Y þ Sbjct: 662 QLVNTTEPVVFISLNGGQMVPVLLDTGSTGLVMDSQFLTQNFGPVIGTGTAGY AGGLTYN 721 Env Score E Sequences producing significant alignments: bits) Value gij15827153jrefjNP_301416.1j hypothetical protein ML0470 [M:::28 0·003 gij15827269jrefjNP_301532.1j putative integral membrane pro:::27 0·006 gij15607880jrefjNP_215254.1j hypothetical protein Rv0740 [M:::27 0·006 gij15609476jrefjNP_216855.1j mmpL9 [Mycobacterium tuberculo:::26 0·007 gij15827153jrefjNP_301416.1jhypotheticalproteinML0470[MycobacteriumlepraeTN] gij2193915jembjCAB09617.1jhypotheticalproteinMLCL581.33c[Mycobacteriumleprae] gij13092701jembjCAC29978.1jhypothetical protein[Mycobacterium leprae] Length ¼ 96 Score ¼ 27·7 bits (60), Expect ¼ 0·003 Identities ¼ 23/75 (30%), Positives ¼ 39/75 (52%), Gaps ¼ 8/75 (10%) Query: 127 VSLKCTDLKNDTNTNSSSGRMIMEKGEIK-NCSFNISTSIRGKVQKEY AFFYKLDIIPID 185 þþKC þ KNSS þ IE þ GE þþSISþ KV K F þþL þ PD Sbjct: 22 ILVKCKSVKQGLNNGSFSRWLINEQGELQWRASLEIS- - -KDKVSK - - - FWFHLTVPND 74 Query: 186 NDTTSYKLTSCNTSV 200 Tþþ þ N þþV Sbjct: 75 AITSANQFYGINSNV 89 gij15827269jrefjNP_301532.1jputative integral membrane protein[Mycobacterium leprae TN] gij13092818jembjCAC30153.1jputative integral membrane protein [Mycobacterium leprae] Length ¼ 983 Score ¼ 26·6 bits (57), Expect ¼ 0·006 Identities ¼ 15/51 (29%), Positives ¼ 26/51 (50%), Gaps ¼ 6/51 (11%) Query: 670 WNWF------NITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQ GYSP 714 WWFþþþþþþþ þ V GLV IVFA L þþRR þ P Sbjct: 43 WLWFGELGYRSVFSTVLVTRVVVFLVAGLVVGGIVFAGLAVAYRTR PVFVP 93 BLASTP 2.0MP-WashU[16-Sep-2002] [decunix4.0-ev6-I32LPF64 2002-09- 18T19:28:12] Copyright (C) 1996–2002 Washington University, Saint Louis, Missouri USA. All Rights Reserved.Reference:Gish, W. (1996–2002) http://blast.wustl.edu Smallest Sum High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N ML0470 hypothetical protein 571603:571893 reverse MW:11046 70 0·0026 1 146 T. Hussain et al. ML0470 hypothetical protein 571603:571893 reverse MW:11046 Full Sequence] [CDS Info] Length ¼ 96 Score ¼ 70 (29·7 bits), Expect ¼ 0·0026, P ¼ 0·0026 Identities ¼ 23/75 (30%), Positives ¼ 37/75 (49%) [HSP Sequence] Query: 127 VSLKCTDLKNDTNTNSSSGGMIMEKGEIK-NCSFNISTSIRGKVQKEY AFFYKHDIIPID 185 þþKC þ KNSS þ IE þ GE þþSISþ KV K F þ H þ PD Sbjct: 22 ILVKCKSVKQGLNNGSFSRWLINEQGELQWRASLEIS- - -KDKVSK- - - - FWFHLTVPND 74 Query: 186 NDTTSYTLTSCNTSV 200 T þþN þþV Sbjct: 75 AITSANQFYGINSNV 89

Appendix 2-VALIDATION AND INTERPRETATION OF THE RESULTS Validation of the results

The anti-IgG internal control band must be present with a strong colour. It is used to validate the addition of the sample and reagents as well as the correct execution of the test protocol. The absence or a low colour intensity of the anti-IgG internal control band indicates either that the sample or reagents were not dispensed or that the test protocol was not followed.

Interpretation of the results A) PROTEINS CONSTITUTIVE OF THE HIV1VIRUS

The presence of anti-HIV1 constitutive proteins antibodies in the samples examined is shown by the appearance of specific coloured bands (blue-violet). Their position corresponds to the molecular masses of the viral proteins listed in the following table.

NAME GENOME NATURE WESTERN BLOT ASPECT

GP 160 ENV , a precursor of GP Clear band 110/120 GP 110/120 ENV Envelope glycoprotein Band with diffuse borders P 68 POL Clear band P55 GAG Precursor of core proteins Doublet P52 POL Reverse transcriptase Clear band GP 41 ENV Transmembrane glycoprotein Diffuse band P 40 GAG Precursor of core proteins Clear band P 34 POL Endonuclease Clear band P 24/25 GAG Core protein Clear band P18 GAG Core protein Sometimes a doublet Cross-reaction with HIV structural proteins 147

INTERPRETATION

Use the positive control to identify the developed antibodies and check that the internal control band is present on each test strip.

INTERPRETATION PROFILE

Positive 2 ENV ^ GAG ^ POL Indeterminate 1 ENV ^ GAG ^ POL; GAG ^ POL; GAG; POL Negative Non-Classified bands No bands

(p) is the level of significance at which the Statistics was found to be significant. For example, P , 0·001 in Table 1 means that there is a significant difference in reactivity to the different HIV structural proteins among the different type of leprosy patients.