Synthetic peptides can be used to develop HIV tests


By Klaus D. Linse 01/04/12

 Antigenic peptides from HIV-1/2 antigenic regions can be used to develop test for the detection of antibodies to HIV-1/2.

Recently researchers located in India report that cocktails made from synthetic antigenic peptides from HIV1/2 antigens can be used for the design of ELISA based tests or assays. Because of the different geographically distribution of different subtypes different specific tests may need to be used in different geographic locations to generate accurate test results. Furthermore the pg 120 protein contains an immunodominant epitope corresponding to the V3 loop that is very hypervariable that can cause different immune reactions in different subtypes.

Infections with the human immunodeficiency virus (HIV) have now become endemic worldwide and AIDS ranks fourth among the world’s top killers of humans. It is estimated that that approximately 34 million people are currently living with one of the ten known subtypes of the HIV infection. Close to 2.7 million people will be newly infected worldwide and an estimated 2.5 million people have already died from this disease. Tiwari et al. in 2013 report that nearly 50% of the newly infected persons belong to the age group of 15–24 years and that the emergence of new variants reflects HIV-1 prevalence, subtype epidemiology, and risk-behavior patterns in different geographical areas. Genetic differences among HIV-1 variants can influence the biological properties of the virus, its susceptibility to existing and candidate anti-retroviral drugs, and evolution of drug resistance.

Strains of HIV-1 can be classified into four groups:

  1. The “major” group M with at least nine genetically distinct subtypes of HIV-1, subtypes A, B, C, D, F, G, H, J, K and CRFs. Subtype A and CRF A/G are predominate in West and Central Africa, with subtype A possibly also causing much of the Russian epidemic. Subtype B has been the most common subtype/CRF in Europe, the Americas, Japan and Australia. Subtype C is common in Southern and East Africa, India and Nepal .
  2. The “outlier” group O, possibly restricted to west-central Africa,
  3. Group N, found in Cameroon, and
  4. Group P.

More info on subtypes can be found at this site: http://www.avert.org/hiv-types.htm

HIV-1 genome map showing the mosaic genome structures of HIV forms

Subtypes

“Mosaic genome structures of the four currently recognized circulating recombinant forms: CRF01_AE, CRF02_AG, CRF03_AB, and CRF04_cpx. An alignment of representative near full-length strains was used. This alignment was gap-stripped prior to all analysis. At the top of the figure, an HIV-1 genome map shows the position of each open reading frame in the gap-stripped multiple alignment. Below the genome map, each bar represents the mosaic pattern of a CRF. The different colours correspond to different subtype assignments. The white regions correspond to unassigned regions. The LTRs were not analyzed. The recombinant regions were inferred from diversity plots, bootscans (Salminen et al. 1995) and when possible from informative sites analysis (Robertson et al. 1995).” (Source: D.L. Robertson, J.P. Anderson, J.A. Bradac, J.K. Carr, B. Foley, R.K. Funkhouser, F. Gao,  B.H. Hahn, M.L. Kalish, C. Kuiken, G.H. Learn, T. Leitner, F. McCutchan, S. Osmanov, M. Peeters, D. Pieniazek, M. Salminen, P. M. Sharp,S. Wolinsky, and B. Korber; HIV-1 Nomenclature Proposal A Reference Guide to HIV-1 Classification).

Tests for HIV

Tests to detect  the human immunodeficiency virus (HIV) that causes the acquired immunodeficiency syndrome (AIDS) are used to detect the virus in blood, serum, saliva, or urine. Most of the tests either detect antibodies, antigens, or RNA molecules corresponding to the virus.

Initially an ELISA based antibody test is performed to test for the presence of HIV or, alternately, a rapid antibody test is used. The newer fourth generation test detects HIV antibodies and antigens simultaneously for both acute and recent HIV infections.  The US Food and Drug Administration has approved a number of HIV test kits for diagnosis, prognostic determination, patient monitoring, and screening of blood and tissue donors. Over the years it has been observed that certain subtypes/CRFs are predominantly associated with the mutations in envelop glycoprotein which allow specific modes of and variations in disease progression. Furthermore, different HIV subtypes can have different immunodominant regions within antigenic proteins that may cause different immune reactions and consequently different antibody titers in the host body. Therefore, the content of specific tests used may need to vary if used in different countries.

Typical test used for HIV testing are:

  1. ELISA based assays or tests (Enzyme-linked immunosorbant assay)
  2. Western blotting
  3. Rapid antibody test as a point-of-care test
  4. Antigen test for the presence of the capsid protein p24
  5. Nucleic acid-based tests

In general nucleic acid-based tests are considered to be highly sensitive and accurate but more time consuming.

Tiwari et al. (2013) used the following peptide cocktail to develop a rapid ELISA based HIV screen assay.

Peptide cocktail used for the development of a HIV screen test

Virus Peptide/Protein Sequence
HIV-1 gp-41 C*RILAVERYLKGQQLLGIWGCSGKLIC*
HIV-2 gp-36 C*QDQARLNSSGCAFRQVC*
HIV-1, Indian isolates V3-I C*TRPNNRKSIRIGBGQTPYATGDIIGDIRGAHC*
HIV-1 rp24 Recombinant protein

The researchers state in their abstract: “A rapid and accurate HIV testing assay is a pre-requisite for practical applicability of diagnostic tests. The aim of this present study was to design a peptide cocktail used as antigens and to develop an ELISA test for HIV-1/2 antibody detection, with enhanced sensitivity and specificity. A novel peptide stretch V3-I, covering immunodominant epitope corresponding to V3 hypervariable loop of gp120 antigens of selected Indian isolates, has been studied and incorporated in an antigenic cocktail of gp36, gp41, and rp24 of HIV-1/2. Peptides from these antigens were chemically synthesized and an additional cysteine residue was added at both amino- and carboxyl-terminal sequences of each peptide in order to form inter and intramolecular disulfide bonds to fold the peptides. This generated conformational epitopes with increased oligomericity and stability of peptide sequences and allowed for better attachment of the antigens to the solid support of ELISA plates. The use of an antigenic cocktail of folded peptides together with the recombinant p24 capsid protein enhanced sensitivity and specificity of the ELISA test. Evaluation of the test using 1123 serum samples in comparison with Boston Biomedical Incorporation (BBI) panels showed 100% sensitivity and 99.3% specificity with no cross reactivity tribulation. In conclusion, “HIV screen test” detects HIV 1/2 antibodies with a high degree of sensitivity and specificity and could be a promising tool for seroscreening of blood during transfusion, counseling and diagnosis of HIV-1/2.

More info on HIV testing can be found at the CDC web site:

http://wwwn.cdc.gov/mpep/hiv-1p24.aspx

HIV virus 2 HIV virus

http://micro.magnet.fsu.edu/cells/viruses/hivvirus.html

http://www.niaid.nih.gov/topics/hivaids/understanding/Pages/Default.aspx

http://www.niaid.nih.gov/topics/hivaids/understanding/biology/Pages/structure.aspx

Info on hypervariable regions of HIV:

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1001228

http://www.uniprot.org/uniprot/Q86720

HIV sequence information:

http://www.ncbi.nlm.nih.gov/nuccore?term=hiv

http://www.ncbi.nlm.nih.gov/genome?term=Human%20immunodeficiency%20virus%201

Reference:

RamP. Tiwari, Anubhav Jain, ZakirKhan, Pradeep Kumar, Vipul Bhrigu, Prakash S. Bisen; Designing of novel antigenic peptide cocktail for the detection of antibodies to HIV-1/2 by ELISA. Journal of Immunological Methods 387 (2013) 157–166.



Categories: Antigens, Artificial Nucleic Acids, Bioanalysis, DNA, DNA Analysis, DNA Editing, DNA finger printing, DNA Structure, H1N1, H7N9, HIV Virus, Human Genetics, Hybridization, Next-generation sequencing, Oligonucleotide Synthesis, peptides, RNA, RNA Editing, RNA FISH, RNA Structure, RNA Synthesis, Sequencing, Vaccines, Virus

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2 replies

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