JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2007, p. 1319–1321 0095-1137/07/$08.00⫹0 doi:10.1128/JCM.01818-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 4
Significant Genetic and Antigenic Variability within the env Gene of Systemic Human Immunodeficiency Virus Type 1 Group O Populations during the Natural Course of a Heterosexual Infection: a Pilot Study䌤 Laurent Andre´oletti,1* Brigitte Re´veil,2 He´le`ne Moret,1 Ve´ronique Brodard,1 Fre´de´rique Philbert,2 Thierry Tabary,2 and Jacques Henri Max Cohen2 Laboratoire de Virologie Ho ˆpital Robert Debre´, IFR 53/EA-3798, CHU et Faculte´ de Me´decine de Reims, Reims, France,1 and Laboratoire d’Immunologie Ho ˆpital Robert Debre´, IFR 53/EA-3798, CHU et Faculte´ de Me´decine de Reims, Reims, France2 Received 1 September 2006/Returned for modification 26 November 2006/Accepted 13 January 2007
We assessed the genetic and the antigenic variability within the env gene of peripheral blood human immunodeficiency virus (HIV) type 1 (HIV-1) group O populations during the natural course of a female heterosexual infection. Our data revealed the existence of a significant increase in amino acid sequence variability within the C2-V3 and gp41 regions (P ⴝ 0.023 and P < 0.001, respectively) in association with substitutions within neutralizing epitope sequences usually selected for HIV serological assays. These antigenic variations might significantly decrease the sensitivity of classical HIV enzyme-linked immunosorbent assays with blood samples of subjects heterosexually infected by HIV-1 group O strains. These findings may be of significant use both to devise diagnostic tools and to pursue suitable therapeutic modalities in cases of heterosexual infection by outlier HIV-1 strains. using the Topo-TA cloning kit (Invitrogen, Groningen, The Netherlands) (2). Nucleotide sequencing was performed with seven subcloned C2-V3 and gp41 PCR products with an ABI 3700 sequencer (Applied Biosystems, Paris, France), as described previously (2). Pairwise distances among nucleotide sequences and amino acid pairwise distances were calculated by using the MEGA 3.1 program (8). Genetic distances between viral clones were compared by analysis of variance test by using Prism Software statistical analysis (version 4; 2003). Coreceptor usage was assessed for each peripheral blood viral strain by infecting indicator cells expressing CD4 and either CXCR4 (373-CXCR4) or CCR5 (373-CXCR5) coreceptor molecules on their surfaces, as described previously (11). The CD4 lymphocyte cell count and HIV viral load were not available for all the study peripheral blood samples. Pairwise nucleotide and amino acid comparisons were computed to assess the genetic variability of the intrapatient HIV group O populations during the natural course of infection. The intrasample nucleotide and amino acid distances, corresponding to the genetic diversity of peripheral blood viral populations, increased significantly between the four study samples (P ⫽ 0.007 and P ⫽ 0.012, respectively) (data not shown). Figure 1A shows the average genetic distance of the sequence of each sample relative to the sequence of the first sample. This distance corresponds to the genetic divergence between the env gene sequences of blood HIV-1 group O populations over the study period. The dynamics of the evolution of the systemic viral populations revealed the existence of a significant increase in amino acid sequence variability within the C2-V3 and the gp41 regions during the 7-year follow-up study (P ⫽ 0.023 and P ⬍ 0.001, respectively). This genetic analysis also revealed the existence of a significantly higher variability within the amino acid sequence of the C2-V3 region than within that of the gp41 region at 36, 72, and 96 months after heterosexual
Aside from epidemiological studies showing a low frequency of human immunodeficiency virus (HIV) group O infections in various populations (1, 10), only a few studies have explored the differences in the genetic and antigenic intrapatient variabilities of group O strains (5, 7, 9). These data are of primary importance to understanding the pathophysiological mechanisms of HIV type 1 (HIV-1) group O infections and are essential for the design of improved diagnostic tests, antiretroviral therapies, and vaccines (10). In the present study, we assessed the genetic and the antigenic variabilities within the env genes of peripheral blood HIV-1 group O populations during the natural course of a heterosexual infection. Moreover, we determined the infectivity and coreceptor usage of peripheral blood HIV-1 group O isolates. We performed a retrospective 7-year follow-up study with sequential peripheral blood samples taken from a treatmentnaive woman heterosexually infected by HIV-1 group O in 1992. Four peripheral blood samples were collected between the time of the initial HIV virological diagnosis (1993) and the presence of clinical signs related to HIV disease (2000), which represents a 7-year period without any antiretroviral therapy. After classical viral RNA extraction from the peripheral blood mononuclear cells, the reverse transcription-PCRs (RT-PCRs) for amplification of a part of the gp120 (C2-V3 region) and the gp41 (immunodominant region) regions of the env genes were performed as described previously (12). For each blood sample, the C2-V3 and gp41 products were each obtained in duplicate from two different RT-PCR runs. For each fragment, the duplicate PCR products were mixed and then cloned by * Corresponding author. Mailing address: Laboratoire de Virologie et EA-3798, Ho ˆpital Robert Debre´, Avenue du Ge´ne´ral Koenig, 51092 Reims Cedex, France. Phone: (33) 3 26 78 39 93. Fax: (33) 3 26 78 41 34. E-mail:
[email protected]. 䌤 Published ahead of print on 31 January 2007. 1319
1320
NOTES
J. CLIN. MICROBIOL.
FIG. 1. Intersample nucleotide and amino acid distances and amino acid sequences in the gp120 C2-V3 and gp41 immunodominant regions of systemic HIV-1 group O variant populations during the natural course of a heterosexual infection. (A) Graphical representation of the average distances of virus sequences from each of the four peripheral blood samples taken during the follow-up study, i.e., between 12 and 96 months after the time of heterosexual infection. Distances were calculated by taking into account the C2V3 or gp41 nucleotide alignment and the derived amino acid sequences from the C2V3 or gp41 regions. Pairwise distances among nucleotide sequences and amino acid pairwise distances were estimated by using the MEGA 3.1 program (8). The average distance between the sequences from each time point was calculated by comparison to the initial C2V3 or gp41 sequences (GenBank accession numbers X84327 and X84328, respectively) and corresponds to the estimated genetic divergence of the two viral env regions. (B) Alignments of amino acid sequences in the gp120 V3 loop and the gp41 immunodominant regions of the systemic HIV-1 variant populations between 12 and 96 months after the time of heterosexual infection. a, after subcloning and sequencing of the PCR products; for the numbers under the number of clones, the numerator represents the number of molecular clones containing identical amino acid sequences and the denominator represents the total number of sequenced molecular clones; b, initial reference sequences of the peripheral blood strain initially published by Cohen et al. (First HIV-O Symp., 1995). This sequence represented the origin reference for the present study; the gp120 amino acids in boldface correspond to the tip of the V3 loop (cysteine residues are labeled by an asterisk to define the limits of the V3 loop), whereas the gp41 sequences in boldface correspond to a major immunodominant peptide previously described (3); c, months postinfection (p.i.), corresponding to the months after the time of suspected heterosexual transmission (considered the minimal time [15 days] before the documented classical clinical signs of HIV primary infection).
infection (P ⬍ 0.001 at each study time) (Fig. 1A). Moreover, we observed several amino acid substitutions within major immunodominant regions (IDRs) located on gp120 (antigenic tip GPGMAWY and its flanking sequences) and on gp41 (R LL—-VQW and LNLWGCKGXXC) of the major viral populations identified (Fig. 1B). HIV cell culture assays of peripheral blood mononuclear cell
samples (11) obtained at each time of the study were positive for reverse transcriptase activity, whereas they were negative for the syncytium-inducing (NSI) biological phenotype (data not shown). All of these HIV-1 group O isolates were able to infect 373 cells expressing CCR5, whereas no virus infection was measured for the 373 cells expressing the CXCR4 coreceptor (data not shown).
VOL. 45, 2007
In the present report, we provide interesting data concerning the genetic and antigenic variabilities of the intrapatient C2-V3 and gp41 env sequences during the natural course of an HIV-1 group O heterosexual infection. The woman from whom the blood samples were obtained has remained clinically asymptomatic with regard to HIV-1 infection during the 7-year follow-up study (CDC stage A2), which is consistent with the absence of the syncytium-inducing biological phenotype observed by the classical cell culture assay of peripheral blood HIV strains (11). This NSI biological phenotype of each HIV strain isolated from that patient correlated with virus infection of 373 cells expressing CCR5 but not CXCR4 on their surfaces (data not shown). The kinetics of the evolution of the viral populations during the follow-up study revealed the existence of a significant increase in genetic variability within two env gene regions, and these increases appeared to be significantly higher within the C2-V3 amino acid sequence than within gp41 amino acid sequence (Fig. 1A). These data are in agreement with those reported by Janssens et al., who described the presence of a high degree of genetic variability within the V3-C3 region of an HIV-1 group O-infected individual during a 10-year follow-up study (7). In the present study, the gp120 genetic variability within the C2-V3 and the gp41 regions appeared to be higher between 12 and 72 months following HIV group O contamination than within the last 12 months of the follow-up study (P ⬍ 0.001 for each of these two env study regions), suggesting that an escape HIV O group mutant emerged within a 6-year period following heterosexual infection (Fig. 1A). The amino acid sequence variability of the two env regions studied appeared to be higher than the nucleotide sequence variability, which corresponded to an excess of nonsynonymous substitutions relative to the number of synonymous substitutions (data not shown). This in itself may be considered evidence of selection for changes in the amino acid sequences of these two HIV group O env gene regions that are involved in different functional and structural roles. These genetic variations must result from various selection pressures (7, 9). Moreover, these env gene regions have previously been identified to contain several epitopes identified as targets of neutralizing antibodies (5, 7, 9, 10). Therefore, our data suggest that the variations within these regions could result from mechanisms of HIV escape from immunological antibody responses (3, 4). Because the tip of the V3 loop as its flanking regions and the two major immunodominant regions located on gp41 (RLL - - - - VQW and LNLWGCKGXXC) (Fig. 1B) can be used as targets to detect the peripheral blood antibodies of patients by classical HIV serological enzyme-linked immunosorbent assays (ELISAs), it might be possible that these antigenic variations may lower the sensitivity of classical HIV serological tests with blood
NOTES
1321
samples after heterosexual infection by HIV-1 group O strains (3, 4). In conclusion, this report reveals the existence of a significant increase in amino acid sequence variability within the C2-V3 and gp41 env gene regions of the peripheral blood viral populations during the natural course of an HIV-1 group O infection by heterosexual contact in a woman. This genetic variability was associated with various substitutions within neutralizing epitope sequences usually selected for by HIV serological assays. These antigenic variations might significantly decrease the sensitivity of classical HIV ELISAs with blood samples of subjects heterosexually infected by HIV-1 group O strains. These findings may be of significant use both to devise diagnostic tools and to pursue suitable therapeutic modalities in cases of heterosexual infection by outlier HIV-1 strains (6, 12). Moreover, the pathogenic consequences of the V3 loop amino acid variations, particularly on disease progression, remain to be elucidated in further, wider prospective national screening programs. Nucleotide sequence accession numbers. The HIV RNA sequences from this study have been deposited in the EMBL database under accession numbers AM262114 to AM262146. Funding for this study was provided by regional grants from the University Hospital of Reims (EA-3798/IFR53). REFERENCES 1. Ayouba, A., P. Mauclere, P. Martin, et al. 2001. HIV-1 group O infection in Cameroon, 1986 to 1998. Emerg. Infect. Dis. 7:466–467. 2. Becquart, P., N. Chomont, P. Roques, et al. 2002. Compartmentalization of HIV-1 between breast milk and blood of HIV-infected mothers. Virology 300:109–117. 3. Dong, X.-N., Y. Wu, and Y.-U. Chen. 2005. The neutralizing epitope ELDKWA on HIV-1 gp41: genetic variability and antigenicity. Immunol. Lett. 101:81–86. 4. Dong, X.-N., Y. Wu, and Y.-U. Chen. 2005. The antigenic tip GPGRAFY of the V3 loop on HIV-1 gp 120: genetic variability and subtypes. Immunol. Lett. 101:112–114. 5. Hunt, J., A. Golden, J. Lund, et al. 1997. Envelope sequence variability and serologic characterization of HIV type 1 group O isolates from Equatorial Guinea. AIDS Res. Hum Retrovir. 13:995–1005. 6. Janssens, W., K. Fransen, I. Loussert-Ajaka, et al. 1995. Diagnosis of HIV-1 group O infection by polymerase chain reaction. Lancet 346:451–452. 7. Janssens, W., J. Nkengasong, L. Heyndrickx, et al. 1999. Intrapatient variability of HIV type I group O ANT70 during a 10-year follow-up. AIDS Res. Hum Retrovir. 15:1325–1332. 8. Kumar S., K. Tamura, and M. Nei. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings Bioinformatics 5:150–163. 9. Loussert-Ajaka, I., M.-L. Chaix, B. Korber, et al. 1995. Variability of human immunodeficiency virus type 1 strains isolated from Cameroonian patients living in France. J. Virol. 69:5640–5649. 10. Quinones-Mateu, M., S. Ball, and E. Arts. 2000. Role of human immunodeficiency virus type 1 group O in the AIDS pandemic. AIDS Rev. 2:190– 202. 11. Trouplin, V., F. Salvatori, F. Cappeloo, et al. 2001. Determination of coreceptor usage of human immunodeficiency virus type 1 from patient plasma samples by using a recombinant phenotypic assay. J. Virol. 75:251–259. 12. Yang, C., B. C. Dash, F. Simon, et al. 2000. Detection of diverse variants of human immunodeficiency virus-1 groups M, N, and O and simian immunodeficiency viruses chimpanzees by using generic pol and env primer pairs. J. Infect. Dis. 18:1791–1795.
