scholarly journals Deep sequencing of the HIV-1 polymerase gene for characterisation of cytotoxic T-lymphocyte epitopes during early and chronic disease stages

2021 ◽  
Author(s):  
Paballo Nkone ◽  
Shayne Loubser ◽  
Thomas C. Quinn ◽  
Andrew D. Redd ◽  
Arshad Ismail ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
Deep Sequencing ◽  
Sanger Sequencing ◽  
Cytotoxic T Lymphocyte ◽  
Sequencing Data ◽  
Subtype C ◽  
Ctl Epitopes ◽  
Significant Difference ◽  
Minority Variant ◽  
Hiv 1

Abstract Background Despite multiple attempts, there is still no effective HIV-1 vaccine available. The HIV-1 polymerase (pol) gene is highly conserved and encodes cytotoxic T-lymphocyte (CTL) epitopes. In this study, deep sequencing was employed for characterisation of HIV-1 Pol CTL epitopes in mostly paired samples obtained during early and chronic stages of infection. Deep sequencing data was then compared to Sanger sequencing data only in samples obtained at baseline. Results Fifty-two participants were enrolled in the study. Their median age was 28 years (interquartile range: 24–32 years) and the majority of participants (92.3%) were female. Illumina minority variant analysis identified a significantly higher number of CTL epitopes (n = 65) compared to epitopes (n = 8) identified through Sanger sequencing. Most of the identified epitopes mapped to reverse transcriptase (RT) and integrase (IN) regardless of the method of sequencing. There was a significantly higher proportion of minority variant epitopes in RT (n = 39, 60.0%) compared to IN (n = 17, 26.2%) and PR (n = 9, 13.8%), p = 0.002 and < 0.0001, respectively. However, no significant difference was observed between the proportion of minority variant epitopes in IN versus PR, p = 0.06. Some epitopes were detected in either early or chronic HIV-1 infection whereas others were detected in both stages. Different distribution patterns of minority variant epitopes were observed in sample pairs; with some increasing or decreasing over time, while others remained constant. Some of the identified epitopes have not been previously reported for HIV-1 subtype C. There were also variants that could not be mapped to reported CTL epitopes in the Los Alamos HIV database. Conclusion Deep sequencing revealed many Pol CTL epitopes, including some not previously reported for HIV-1 subtype C. The findings of this study support the inclusion of RT and IN epitopes in HIV-1 vaccine candidates as these proteins harbour many CTL epitopes. Variants that were not mapped within CTL epitopes could represent new epitopes.

scholarly journals Deep Sequencing of the HIV-1 Polymerase Gene for Characterisation of Cytotoxic T-Lymphocyte Epitopes During Early and Chronic Disease Stages

2021 ◽  
Author(s):  
Paballo Nkone ◽  
Shayne Loubser ◽  
Thomas C. Quinn ◽  
Andrew D. Redd ◽  
Arshad Ismail ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
Deep Sequencing ◽  
Sanger Sequencing ◽  
Cytotoxic T Lymphocyte ◽  
Subtype C ◽  
Los Alamos ◽  
Ctl Epitopes ◽  
Significant Difference ◽  
Minority Variant ◽  
Hiv 1

Abstract Background Despite multiple attempts, there is still no effective HIV-1 vaccine available. The HIV-1 polymerase (pol) gene is highly conserved and encodes cytotoxic T-lymphocyte (CTL) epitopes. The aim of the study was to characterise HIV-1 Pol CTL epitopes in mostly sample pairs obtained during early and chronic stages of infection. Methods Illumina deep sequencing was conducted for all samples while Sanger sequencing was only performed on baseline samples. Codons under immune selection pressure were assessed by computing nonsynonymous to synonymous mutation ratios using MEGA. Minority CTL epitope variants occurring at \(\ge\)5% were detected using low-frequency variant tool in CLC Genomics. Los Alamos HIV database was used for mapping mutations to known HIV-1 CTL epitopes. Results Fifty-two participants were enrolled in the study. Their median age was 28 years (interquartile range: 24–32 years) and majority of participants (92.3%) were female. Illumina minority variant analysis identified a significantly higher number of CTL epitopes (n = 65) compared to epitopes (n = 8) identified through Sanger sequencing. Most of the identified epitopes mapped to reverse transcriptase (RT) and integrase (IN) regardless of sequencing method. There was a significantly higher proportion of minority variant epitopes in RT (n = 39, 60.0%) compared to IN (n = 17, 26.2%) and PR (n = 9, 13.8%), p = 0.002 and < 0.0001, respectively. However, no significant difference was observed between the proportion of minority variant epitopes in IN versus PR, p = 0.06. Some epitopes were detected in either early or chronic HIV-1 infection whereas others were detected in both stages. Different distribution patterns of minority variant epitopes were observed in sample pairs; with some increasing or decreasing over time, while others remained constant. Some of the identified epitopes have not been previously reported for HIV-1 subtype C. There were also variants that could not be mapped to reported CTL epitopes in the Los Alamos HIV database. Conclusion Deep sequencing revealed many Pol CTL epitopes, including some not previously reported for HIV-1 subtype C. The findings of this study support the inclusion of RT and IN epitopes in HIV-1 vaccine candidates as these proteins harbour many CTL epitopes.

scholarly journals Identification of Dominant Optimal HLA-B60- and HLA-B61-Restricted Cytotoxic T-Lymphocyte (CTL) Epitopes: Rapid Characterization of CTL Responses by Enzyme-Linked Immunospot Assay

2000 ◽  
Vol 74 (18) ◽  
pp. 8541-8549 ◽  
Author(s):  
Marcus A. Altfeld ◽  
Alicja Trocha ◽  
Robert L. Eldridge ◽  
Eric S. Rosenberg ◽  
Mary N. Phillips ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
Mononuclear Cells ◽  
Cytotoxic T Lymphocyte ◽  
Hla Class I ◽  
Peripheral Blood Mononuclear ◽  
Antiviral Immune Response ◽  
Ctl Epitopes ◽  
Hla Class I Molecules ◽  
Hiv 1 ◽  
Ctl Responses

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1.

scholarly journals Between-Host Evolution of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1: an Approach Based on Phylogenetically Independent Comparisons

2004 ◽  
Vol 78 (21) ◽  
pp. 11758-11765 ◽  
Author(s):  
Helen Piontkivska ◽  
Austin L. Hughes
Keyword(s):  
Natural Selection ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Ctl Epitopes ◽  
Term Evolution ◽  
Immunodeficiency Virus ◽  
Host Evolution ◽  
Hiv 1

ABSTRACT In human immunodeficiency virus type 1 (HIV-1), mutations that escape from cytotoxic T-lymphocyte (CTL) recognition have been documented, and sequence analyses have provided indirect support for the hypothesis that natural selection has favored CTL escape mutants within an infected host. In spite of such evidence for within-host selection by CTL, it has been more difficult to determine how natural selection by host CTL has influenced long-term evolution of HIV-1. We used statistical analysis of published HIV-1 genomic sequences to examine the role of natural selection in between-host evolution of CTL epitopes. Based on a phylogenetic analysis, we identified 21 pairs of closely related genomes isolated from different hosts and examined the pattern of nucleotide substitution in genomic regions encoding well-characterized CTL epitopes. The results revealed that certain CTL epitopes have been subject to repeated positive selection across the population, while others are generally conserved. Furthermore, evidence of positive selection was associated with divergence from the canonical epitope sequence and with an enhanced frequency of convergent amino acid sequence changes in CTL epitopes. The results support the hypothesis that CTL-driven selection has been a major factor in the long-term evolution of HIV-1.

scholarly journals Immune Selection for Altered Antigen Processing Leads to Cytotoxic T Lymphocyte Escape in Chronic HIV-1 Infection

2004 ◽  
Vol 199 (7) ◽  
pp. 905-915 ◽  
Author(s):  
Rika Draenert ◽  
Sylvie Le Gall ◽  
Katja J. Pfafferott ◽  
Alasdair J. Leslie ◽  
Polan Chetty ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
Antigen Processing ◽  
Cytotoxic T Lymphocyte ◽  
Immune Selection ◽  
Ctl Epitopes ◽  
Leukocyte Antigen ◽  
Human Infections ◽  
Flanking Regions ◽  
Hiv 1 ◽  
Restricted Epitope

Mutations within cytotoxic T lymphocyte (CTL) epitopes impair T cell recognition, but escape mutations arising in flanking regions that alter antigen processing have not been defined in natural human infections. In human histocompatibility leukocyte antigen (HLA)-B57+ HIV-infected persons, immune selection pressure leads to a mutation from alanine to proline at Gag residue 146 immediately preceding the NH2 terminus of a dominant HLA-B57–restricted epitope, ISPRTLNAW. Although N-extended wild-type or mutant peptides remained well-recognized, mutant virus–infected CD4 T cells failed to be recognized by the same CTL clones. The A146P mutation prevented NH2-terminal trimming of the optimal epitope by the endoplasmic reticulum aminopeptidase I. These results demonstrate that allele-associated sequence variation within the flanking region of CTL epitopes can alter antigen processing. Identifying such mutations is of major relevance in the construction of vaccine sequences.

scholarly journals Clustering Patterns of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1 (HIV-1) Proteins Reveal Imprints of Immune Evasion on HIV-1 Global Variation

2002 ◽  
Vol 76 (17) ◽  
pp. 8757-8768 ◽  
Author(s):  
Karina Yusim ◽  
Can Kesmir ◽  
Brian Gaschen ◽  
Marylyn M. Addo ◽  
Marcus Altfeld ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Amino Acid Sequences ◽  
Ctl Epitopes ◽  
Ctl Epitope ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequences reveal that defined epitopes tend to cluster. Here we integrate the global sequence and immunology databases to systematically explore the relationship between HIV-1 amino acid sequences and CTL epitope distributions. CTL responses to five HIV-1 proteins, Gag p17, Gag p24, reverse transcriptase (RT), Env, and Nef, have been particularly well characterized in the literature to date. Through comparing CTL epitope distributions in these five proteins to global protein sequence alignments, we identified distinct characteristics of HIV amino acid sequences that correlate with CTL epitope localization. First, experimentally defined HIV CTL epitopes are concentrated in relatively conserved regions. Second, the highly variable regions that lack epitopes bear cumulative evidence of past immune escape that may make them relatively refractive to CTLs: a paucity of predicted proteasome processing sites and an enrichment for amino acids that do not serve as C-terminal anchor residues. Finally, CTL epitopes are more highly concentrated in alpha-helical regions of proteins. Based on amino acid sequence characteristics, in a blinded fashion, we predicted regions in HIV regulatory and accessory proteins that would be likely to contain CTL epitopes; these predictions were then validated by comparison to new sets of experimentally defined epitopes in HIV-1 Rev, Tat, Vif, and Vpr.

scholarly journals Differences in Evolution of HIV-1 Subtype C Reverse Transcriptase Between Children and Adults Likely Explained by Maturity of Cytotoxic T-Lymphocyte Responses

2015 ◽  
Vol 31 (6) ◽  
pp. 655-657
Author(s):  
Shanmugam Saravanan ◽  
Selvamurthi Gomathi ◽  
Bagavathi Kausalya ◽  
Jayaseelan Boobalan ◽  
Kailapuri G Murugavel ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Subtype C ◽  
Lymphocyte Responses ◽  
Hiv 1

scholarly journals Magnitude and Frequency of Cytotoxic T-Lymphocyte Responses: Identification of Immunodominant Regions of Human Immunodeficiency Virus Type 1 Subtype C

2002 ◽  
Vol 76 (20) ◽  
pp. 10155-10168 ◽  
Author(s):  
V. Novitsky ◽  
H. Cao ◽  
N. Rybak ◽  
P. Gilbert ◽  
M. F. McLane ◽  
...  
Keyword(s):  
Immune Responses ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Population Level ◽  
Hiv Vaccine ◽  
Subtype C ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Ctl Responses

ABSTRACT A systematic analysis of immune responses on a population level is critical for a human immunodeficiency virus type 1 (HIV-1) vaccine design. Our studies in Botswana on (i) molecular analysis of the HIV-1 subtype C (HIV-1C) epidemic, (ii) frequencies of major histocompatibility complex class I HLA types, and (iii) cytotoxic T-lymphocyte (CTL) responses in the course of natural infection allowed us to address HIV-1C-specific immune responses on a population level. We analyzed the magnitude and frequency of the gamma interferon ELISPOT-based CTL responses and translated them into normalized cumulative CTL responses. The introduction of population-based cumulative CTL responses reflected both (i) essentials of the predominant virus circulating locally in Botswana and (ii) specificities of the genetic background of the Botswana population, and it allowed the identification of immunodominant regions across the entire HIV-1C. The most robust and vigorous immune responses were found within the HIV-1C proteins Gag p24, Vpr, Tat, and Nef. In addition, moderately strong responses were scattered across Gag p24, Pol reverse transcriptase and integrase, Vif, Tat, Env gp120 and gp41, and Nef. Assuming that at least some of the immune responses are protective, these identified immunodominant regions could be utilized in designing an HIV vaccine candidate for the population of southern Africa. Targeting multiple immunodominant regions should improve the overall vaccine immunogenicity in the local population and minimize viral escape from immune recognition. Furthermore, the analysis of HIV-1C-specific immune responses on a population level represents a comprehensive systematic approach in HIV vaccine design and should be considered for other HIV-1 subtypes and/or different geographic areas.

scholarly journals Molecular Characteristics of Human Immunodeficiency Virus Type 1 Subtype C Viruses from KwaZulu-Natal, South Africa: Implications for Vaccine and Antiretroviral Control Strategies

2003 ◽  
Vol 77 (4) ◽  
pp. 2587-2599 ◽  
Author(s):  
M. Gordon ◽  
T. De Oliveira ◽  
K. Bishop ◽  
H. M. Coovadia ◽  
L. Madurai ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Reverse Transcriptase ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Control Strategies ◽  
Subtype C ◽  
Kwazulu Natal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of human immunodeficiency virus type 1 (HIV-1) subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or “lineages” of HIV-1 subtype C that segregated with other C viruses from southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.

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