The introduction of combined ART has dramatically changed the course of infections with the human immunodeficiency virus type 1 (HIV-1) in adults and children . In current guidelines ART is recommended for all HIV-1-infected infants in the first year of life regardless of the HIV clinical, immunological and virological category, because infants starting ART in the first year of life have less progression to AIDS compared with those starting later . Treatment of HIV-1-infected children under 2 years of age is challenging due to the limited availability of appropriate drugs, poor palatability of liquid formulations, difficulties in therapy adherence and long-term ART-toxicity making full viral suppression difficult and a rebound of viral replication more frequent than in adults .
In infants starting ART in the first months of life and achieving effective virus suppression HIV-1 seroreversion has been documented in a subset of patients [4,5]. We reported on a child initially presenting with AIDS who achieved HIV-1 seroreversion following effective ART . It has been postulated that HIV-1 seroreversion might not be an exception when infants are treated early and efficiently . However, in clinical practice seroreversion is observed only in few infants, whereas most infants develop and maintain a robust antibody response to the infection despite early therapy, effective virus suppression and good therapy adherence. The physiological requirements for seroreversion in infants receiving ART are not clearly understood. Besides external factors for example maternal antibodies and virus subtype, the difference in antibody response between seroreverted and nonseroreverted infants might be caused by genetic determinants.
Host genetic factors were reported to fundamentally determine HIV-1 susceptibility and disease progression [8,9]. During immune response against HIV-1 many of them directly or indirectly participate in virus recognition (chemokine receptors, human leukocyte antigen (HLA), T-cell receptor (TCR), killer immunoglobulin-like receptors (KIRs), and toll-like receptors (TLRs)) [8,9]. A homozygous 32 bp deletion in the chemokine receptor CCR5 has long been identified as protective variant against HIV-1 infection . As another determinant, heterozygosity at HLA class I regions appears to provide an advantage for the immune system against the development of AIDS, because it expands the hosts ability to present viral antigens and thereby broadens the immune response. In addition, various individual HLA class I and II alleles have been associated with either slow (e.g. HLA-B*57, B*3502, B*3503, B*3504, and DRB1*13) or fast disease progression (e.g. HLA-B*3501 and B*3508) following HIV-1 infection [8,9].
The aim of this study was to follow-up patients with initially documented HIV-1 seroreversion receiving early ART and to examine whether, in addition to early and efficient treatment, HIV-1 seroreversion in HIV-1-infected children might also depend on genetic determinants.
Patients with HIV-1 seroreversion were identified using a standardized questionnaire that was sent to clinicians caring for children with HIV-1 infection in Germany. Patients were enrolled in the study, if they had at least one negative HIV-1 antibody test result following effective ART in their medical history. Eight seronegative patients were enrolled from five sites between 2011 and 2012. HIV-1-infected children who started therapy before 5 months of age but seroconverted despite effective viral suppression served as controls for genetic analysis. Research was conducted in accordance with the Declaration of Helsinki, revised in 2008. The study was approved by the ethics committee of the Medical Faculty of the Heinrich Heine University Düsseldorf. All parents gave written informed consent prior to enrollment.
From each patient 3–12 ml EDTA-anticoagulated peripheral blood was obtained. Lymphocyte subsets were characterized on whole blood by standard flow cytometry. Cells were stained with monoclonal antibodies to CD3 [SK7, Becton Dickinson GmbH (BD), Germany], CD4 (SK3, BD), CD8 (SK1, BD), CD25 (B149.9, Beckmann Coulter GmbH [BC], Germany), CD45RA (L48, BD), CD45RO (UCLH1, BD) and HLA-DR (L243, BD) for T-cell subsets, CD19 (SJ25C1, BD), CD20 (L27, BD), CD27 (1A4, BC) and IgD (IA62, BD) for B-cell subsets and, additionally, CD56 (My31, BD) for NK cell identification.
From each sample 400 μl plasma was obtained by standard procedures. Expression of antibodies targeting HIV-1 proteins was monitored at study entry using INNO-LIA HIV I/II Score (Innogenetics N.V., Belgium). PBMC were isolated form peripheral blood by standard Ficoll-Paque density gradient centrifugation. DNA from the naive CD4+ and CD8+ subsets was isolated by anion exchange chromatography according to the manufacturer (AllPrep DNA/RNA/Protein Mini Kit; Qiagen GmbH, Germany).
Genetic analyses were performed on DNA pooled from naive CD4 and CD8 fractions. CCR5 genotypes were initially characterized by PCR using published primers  and confirmed by direct sequencing (BigDye Terminator v1.1 Cycle Sequencing Kit; Life Technologies GmbH, Germany). Genotypes were further validated using allele-specific PCR primers (CCR5 wt: 5′-TCATTTTCCATACAGTCAGT, CCR5 Δ32: 5′-TCATTTTCCATACATTAAAG; Eurofins MWG Operon, Germany).
HLA-genotyping of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA0-DQB1 was performed using SBT excellarator HLA-kits (GenDX, Netherlands). Genomic DNA was amplified using primers flanking exons 2–4 (HLA-A, HLA-B,HLA-C) or exon 2 (HLA-DRB1, HLA-DQB1). PCR products were purified by ExoSAP-IT (Affymetrix, USA). Remaining dyes of the sequencing reactions were removed using the BigDye XTerminator Purification Kit (Applied Biosystems, USA). Sequencing reactions were analyzed on a 3730 DNA Analyzer (Applied Biosystems).
Thirty-seven children who had started therapy before 5 months of age and achieved effective virus suppression following ART were reported from the participating centres. Eleven of these children had documented HIV-1 seroreversion and eight were recruited for our study. The patient characteristics are shown in Table 1. Mean age at follow-up was 6.6 ± 3.9 years. Effective prenatal screening and prophylaxis to prevent mother to child HIV-1 transmission especially antiretroviral treatment of mothers was missing in all eight patients.
Five out of eight children still had negative HIV-1 antibody tests at study entry (Table 1). Although two patients showed partial reactivity against p24, this is interpreted as negative assay result in standard diagnostic testing . Three out of eight children had developed an antibody response against HIV-1 resulting in positive antibody tests at the time point of follow-up. Only one of these patients had a complete reactivity against all tested HIV epitopes (P3). This child had experienced treatment failure due to adherence problems and therapy interruption but returned to undetectable viral load after switching ART. The remaining two patients with a positive HIV-1 antibody test (P2 and P2) showed only partial reactivity. Except for the patient who interrupted ART, neither of the patients evolved antibodies against gp120 and only two generated antibodies against gp41.
All patients had normal numbers and frequencies of overall lymphocytes as well as T cells, B cells and NK cells (Table 1) with only negligible deviations from standard values of age-matched healthy children . With regard to the vulnerable T-cell compartment, no decrease of naive CD4+ T cells was observed. Two patients (P1 and P3) showed an increase in the naive CD4 pool. One patient (P2) had an expansion in the memory CD8+ T-cell pool; however, this patient also presented increased numbers of naive CD8+ cells.
Sequencing of the CCR5 coding region revealed no deviation from the reference sequence with exception of patient P3 featuring a heterozygous form of the Δ32 variant (Table 2). The results of the high-resolution HLA genotyping are shown in Table 2. The most intriguing association was found for HLA class II locus DQB1. We found seven out of eight patients carrying a *03 allele and four of those patients additionally harbored a *06 allele. Additionally, three of the patients positive for DQB1*03 and DQB1*06 also carried DRB1*13, an allele which had already been implicated in slow disease progression .
Although it is quite challenging to regularly administer antiretroviral treatment to infants, our data show that, in a subset of patients, HIV-1 replication can be suppressed to an extent that the mounted humoral immune response does not result in antibody generation against HIV proteins (HIV seroreversion). Maintenance of HIV-1 seroreversion following ART is not limited to infancy, because the oldest patient demonstrating continuous seronegativity following ART in our cohort was 12 years old. However, since a large proportion of HIV-1-infected infants experience seroconversion despite early and efficient reduction of viral RNA loads, the seroreversion phenotype appears to depend on crucial factors.
In our study, we focused on genetic determinants that could account for the distinct development of seronegative and seropositive phenotypes after early initiation of ART. Analysis of the CCR5 genotype revealed only one patient harboring a heterozygous CCR5 Δ32 allele indicating that the chemokine receptor is not involved in HIV-1 seroreversion in infants. A much more intriguing association was found for the HLA class II region with seven out of eight patients carrying a DQB1*03 allele. Three out of four seroreverted patients from Africa or Europe carried the allele DQB1*03:SAK (allele frequency = 0.375). The DQB1*03:SAK frequency is 0.096–0356 in Africa and 0.033–0349 in Europe . As this allele has so far not been correlated with modulation of disease progression, our finding might indicate a new role for DQB1*03:SAK in the immune response against HIV-1. Furthermore, four out of eight patients carried a DQB1*06 allele, three of them in combination with DRB1*13. This DRB1 allele has already been associated with long-term survival among children with vertically transmitted HIV-1 infection . In addition, the DRB1*13-DQB1*06 haplotype was linked to a trend towards increased AIDS-free time following HIV infection in adults  and patients who inherited HLA-DRB1*13-DQB1*06 had a greater likelihood of controlling HIV-1 replication and maintaining T-cell help activities . Three out of eight (38%) patients in our cohort carried HLA-DRB1*13-DQB1*06 indicating a potential cooperative effect of both class II loci in generating the HIV-1 seroreversion phenotype. The importance of HLA class II alleles might relate to the central role of CD4+ T cells in sustaining cytotoxic T-cell and B-cell activation, which depends on the recognition of HIV-1 epitopes presented by MHC class II.
Increased heterozygosity at HLA class I regions is thought to be an advantage for the immune system against AIDS, because it broadens the repertoire of presented viral antigens. Twenty-eight to 40% of HIV-1-infected Caucasian patients who were able to delay AIDS for 10 or more years were fully heterozygous at all HLA class I loci . In our cohort, six to eight patients were heterozygous at all loci. However, the majority of control patients was also fully heterozygous at all HLA loci. Therefore, HLA heterozygosity is not decisive for HIV-1 seroreversion. HLA alleles that have been associated with slow disease progression were found in our cohort, but none of the known dominant protective HLA alleles (e.g. B*27 and B*57) were enriched in our study population.
From our data, it might be suggested that HLA-DQB1*03 especially DQB1*03:SAK and DQB1*06 could support efficient clearing of viral antigens mediated by early and efficient ART. However, the size of the patient cohort is still limited. As treatment options for infants are improving, the number of patients with HIV-1 seroreversion is expected to increase and thus this correlation might be tested in a larger study cohort.
We would like to thank the patients and their families, who participated in this study. We thank E. Oellers for excellent technical assistance.
Authors contribution: C.A. (biologist): conception and design of the study, sample processing, CCR5 PCR, collection of data, analysis and interpretation of data, drafting of the article.
J.N. (pediatrician): conception and design of the study, coordination of the study, acquisition of pediatric data, analysis and interpretation of data, drafting of the article.
O.A. (virologist): performed the virologic assays at study entry, interpretation of virologic data, critical revision of the article, approved the final version of the article.
H.-J.L. (pediatrician): acquisition of pediatric data, analysis and interpretation of data, critical revision of the article, approved the final version of the article.
C.F.S. (pediatrician): acquisition of pediatric data, interpretation of data, critical revision of the article, approved the final version of the article.
B.B. (pediatrician) acquisition of pediatric data, interpretation of data, critical revision of the article, approved the final version of the article.
G.N. (pediatrician) acquisition of pediatric data, interpretation of data, critical revision of the article, approved the final version of the article.
J.E.: HLA typing, interpretation of genetic data, critical revision of the article, approved the final version of the article.
A.B. (pediatrician, Head of Department): conception and design of the study, analysis and interpretation of data, critical revision of the article, approved the final version.
Conflicts of interest
There are no conflicts of interest.
1. Gortmaker SL, Hughes M, Cervia J, Brady M, Johnson GM, Seage GR 3rd, et al. Effect of combination therapy including protease inhibitors on mortality among children and adolescents infected with HIV-1
. N Engl J Med
2. Violari A, Cotton MF, Gibb DM, Babiker AG, Steyn J, Madhi SA, et al. Early antiretroviral therapy and mortality among HIV-infected infants
. N Engl J Med
3. van Rossum AM, Fraaij PL, de Groot R. Efficacy of highly active antiretroviral therapy in HIV-1 infected children
. Lancet Infect Dis
4. Hainaut M, Peltier CA, Gerard M, Marissens D, Zissis G, Levy J. Effectiveness of antiretroviral therapy initiated before the age of 2 months in infants vertically infected with human immunodeficiency virus type 1
. Eur J Pediatr
5. Luzuriaga K, McManus M, Catalina M, Mayack S, Sharkey M, Stevenson M, et al. Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses
. J Virol
6. Neubert J, Laws HJ, Adams O, Munk C, Kramer M, Niehues T, et al. HIV-1 seroreversion following antiretroviral therapy in an HIV-infected child initially presenting with acquired immunodeficiency syndrome
7. Eberle J, Notheis G, Blattmann C, Jung J, Buchholz B, Korn K, et al. Seroreversion in vertically HIV-1-infected children treated early and efficiently: rule or exception?
8. Singh KK, Spector SA. Host genetic determinants of human immunodeficiency virus infection and disease progression in children
. Pediatr Res
9. Kaur G, Mehra N. Genetic determinants of HIV-1 infection and progression to AIDS: immune response genes
. Tissue Antigens
10. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection
11. Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation
. N Engl J Med
12. Centers for Disease ControlInterpretation and use of the western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections
13. van Gent R, van Tilburg CM, Nibbelke EE, Otto SA, Gaiser JF, Janssens-Korpela PL, et al. Refined characterization and reference values of the pediatric T- and B-cell compartments
. Clin Immunol
14. Chen Y, Winchester R, Korber B, Gagliano J, Bryson Y, Hutto C, et al. Influence of HLA alleles on the rate of progression of vertically transmitted HIV infection in children: association of several HLA-DR13 alleles with long-term survivorship and the potential association of HLA-A*2301 with rapid progression to AIDS. Long-Term Survivor Study
. Hum Immunol
15. Gonzalez-Galarza FF, Christmas S, Middleton D, Jones AR. Allele frequency net: a database and online repository for immune gene frequencies in worldwide populations
. Nucleic Acids Res
16. Keet IP, Tang J, Klein MR, LeBlanc S, Enger C, Rivers C, et al. Consistent associations of HLA class I and II and transporter gene products with progression of human immunodeficiency virus type 1 infection in homosexual men
. J Infect Dis
17. Malhotra U, Holte S, Dutta S, Berrey MM, Delpit E, Koelle DM, et al. Role for HLA class II molecules in HIV-1 suppression and cellular immunity following antiretroviral treatment
. J Clin Invest
18. Carrington M, Nelson GW, Martin MP, Kissner T, Vlahov D, Goedert JJ, et al. HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage