Occult hepatitis B virus infection and S gene escape mutants in HIV-infected patients after hepatitis B virus vaccination

Abstract

Hepatitis B virus (HBV) vaccination is recommended for HIV patients. Despite the relative success of HBV vaccination, breakthrough infections can occur infrequently in patients, and it can be due to occult HBV infection, vaccine unresponsiveness and/or emergence of escape mutants. This study assessed the presence of occult HBV infection and S gene escape mutants in HIV-positive patients after HBV vaccination. Ninety-two HIV-positive patients were enrolled in this study, including 52 responders to HBV vaccine and 40 non-responders. All of the cases received HBV vaccine according to routine HBV vaccination protocols. The presence of HBV-DNA was determined by real-time polymerase chain reaction (PCR). In HBV-DNA positive samples, the most conserved regions of S gene sequences were amplified by nested PCR and PCR products were sequenced. Occult HBV infection was detected in two cases. Glycine to arginine mutation at residue 145 (G145R) within the ‘a’ region of the S gene was detected in one of the occult HBV infection cases who was in the non-responder group. This study showed that the prevalence of occult HBV infection and vaccine escape mutants was low in our HBV-vaccinated HIV-positive patients in both responder and non-responder groups, so there was no alarming evidence indicating breakthrough HBV infection in our vaccinated HIV-positive cases.

Keywords

Human immunodeficiency virus HIV AIDS hepatitis B virus occult hepatitis B virus infection S gene mutation vaccination

Introduction

Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is common, due to similar routes of transmission.¹ In HBV low endemicity countries, the HBV co-infection is about 5–7% in HIV-infected patients.² In intermediate and high HBV endemicity areas, HBV/HIV co-infection rate is about 10–20%.^3,4

HIV-infected patients have higher levels of hepatitis B viremia, rapidly progressing to chronic hepatitis B and higher risk of cirrhosis and hepatocellular carcinoma.⁵ Some studies showed that HBV infection caused faster progression of HIV infection to AIDS, by increasing the expression of HIV-infected cells and more rapid decrease in CD4 lymphocytes,^6,7 but the effects of HBV on the progression of HIV disease are controversial. Therefore, HBV vaccination is highly recommended to all HIV-infected patients without evidence of prior HBV infection or immunity to HBV according to the current guidelines.^8,9 On the other hand, HIV can cause poorer antibody response to HBV vaccination. The immunogenicity of HBV vaccination in HIV patients varies from 17.5% to 72%.¹⁰

Despite the relative success of HBV vaccination in HIV patients, breakthrough infections can occur infrequently in these patients, and it can be due to occult HBV infection (OBI), vaccine unresponsiveness and/or emergence of escape mutants.^11,12

OBI is defined as the presence of HBV-DNA in the liver or serum with undetectable hepatitis B surface antigen (HBsAg).¹³ The emergence of S gene variants, with mutations occurring mainly within the ‘a’ determinant region located among amino acids 124–147 of hepatitis B surface antigen, has been observed commonly in persons who had been vaccinated in several regions of the world.^14–16 These mutations are characterised by a glycine to arginine mutation at residue 145 (G145R) within the ‘a’ determinant region, due to a G to A transition at nucleotide position 587.^17,18 Most of the breakthrough infections are related to wild-type HBV, but mutants in the antigenic ‘a’ determinant are found in approximately 20% of infected subjects.^19–21

Some studies investigated prevalence of OBI and HBV S gene mutations in some groups after HBV vaccination,^22,23 but few studies have been conducted regarding these data in HIV patients. So the aim of this study was to assess the presence of OBI and S gene escape mutants in HIV-positive patients after HBV vaccination.

Patients and methods

Study population

In this cross-sectional study, 92 HIV-positive patients including 52 responders to HBV vaccine and 40 non-responders from the Iranian Research Center for HIV/AIDS, Tehran, Iran were enrolled from March to December 2014. All of the cases had received HBV vaccine according to the routine HBV vaccination protocol (three intramuscular injections of the standard dose [20 µg] of recombinant HBV vaccine [Pasteur Institute of Iran, Tehran, Iran] at months 0, 1, 6) not more than a year ago. All cases had never received HBV vaccination before this study. A protective antibody response was defined as being a hepatitis B surface antibody (anti-HBs) titre ≥10 IU/L.

This project was approved by the Pasteur Institute of Iran ethics committee and informed consent was obtained from patients prior to their enrollment. A questionnaire that gathered epidemiological, clinical and laboratory data was completed by the clinicians.

Serological tests

Human immunodeficiency virus antibody (anti-HIV) was determined by ELISA (MP Biomedicals, Illkirch, France), with positive tests confirmed by Western blot assay (Diaplus, San Francisco, USA). All samples were tested for HBsAg, anti-HBs, hepatitis B core antibody (anti-HBc) and anti-HCV by enzyme-linked immunosorbent assay (ELISA). The commercial enzyme immunoassay kits used were as follows: HBsAg (Hepanosticka Biomerieux, Boxtel, The Netherlands), anti-HBs (Enzygnost, Dade Behring Marburg GmbH, Germany), anti-HBc (Enzygnost, Dade Behring Marburg GmbH, Germany) and anti-HCV (Biorad, Segrate, Italy). Recombinant immunoblot assay (RIBA Innogenetics, Ghent, Belgium) was employed to confirm anti-HCV reactivity.

DNA extraction and real-time polymerase chain reaction

HBV-DNA was extracted using Qiamp DNA Mini DNA (QIAGEN, USA) following the manufacturer’s instructions. HBV-DNA was determined by real-time polymerase chain reaction (PCR) using the Real Star HBV PCR kit (altona Diagnostics GmbH, Germany) on the Rotor-Gene 6000 real-time thermal cycler (Corbett Research, Sydney, Australia). The detection limit of the kit is 50 IU/mL according to the user manual.

Amplification of HBV S-ORF and sequencing assay

The most conserved regions of S gene sequences were amplified by nested PCR, using the primers S1A (5′-CCTGCTGGTGGCTCCAGTTC-3′) and antisense (′5-CCACAATTCKTTGACATACTTTCCA-3′). Nested PCR was carried out to increase the sensitivity of detection and to provide an amplicon (712 bp) for sequence analysis using the primers sense (5′-GAGAATTCCGAGGACTGGGGACCCTG-3′) and S2 (5′-CGGGATCCT TAGGGTTTAAATGTATACC-3′). A 10-µL aliquot of extracted DNA was added to an amplification mixture containing 5 µL of 10× PCR buffer, 1.5 µL of MgCl₂ (50 mM), 1 µL of dNTP mix (100 mM each), 1 µL (2.5 U) of Taq polymerase (CinnaGen, Tehran, Iran) and 2 µL of each of the outer primers (10 pmol) in a total volume of 50 µL. The PCR profile was an initial 3-min denaturation at 94, followed by 35 cycles of amplification including denaturation for 30 s at 94, annealing for 30 s at 59 and extension for 40 s at 72. Strand synthesis was completed at 72 for 6 min. 2 µL of the first-round PCR product was then subjected to a second-round PCR under the same conditions but at an annealing temperature of 62. Second-round PCR products were subjected to bidirectional automated sequencing using both forward and reverse inner primers (Pasteur Institute of Iran, Tehran, Iran).

Nucleotide sequences were aligned with the CLUSTALW program using BioEdit software (BioEdit Sequence Alignment Editor Software, Department of Microbiology, North California State University). Variants were compared with original sequences of this genotype for identifying mutations.

Statistical analysis

The Chi square test was used with the SPSS 16 program for statistical analysis (Chicago, IL, USA). Data are presented as mean ± SD or, when indicated, as an absolute number and percentage.

Results

In this study, 92 HIV-infected patients were enrolled. The mean age of patients was 37.97 ± 8.11 years; 54.3% of patients were men and 45.7% were women. The mean CD4 count of HIV subjects was 424 ± 226 cells/mm³. The presumed routes of HIV transmission were heterosexual contact (48.9%), intravenous drug use (41.3%), infected blood and blood products transfusion (3.3%), tattooing (1.1%) and in 5.4% the route of HIV acquisition was not identified. All of the patients received antiretroviral therapy (ART) (combination of AZT [zidovudine], 3TC [lamivudine] and EFV [efavirenz] in 90% of patients and the other cases received other ART including Kaletra [lopinavir/ritonavir], Truvada [tenofovir/emtricitabine], tenofovir and nevirapine) with a mean duration of 35.2 ± 21.8 months. HBsAg and anti-HBc were negative in all cases; 33% of cases were anti-HCV positive.

All of the cases had received HBV vaccine according to the routine HBV vaccination protocol not more than a year ago. A protective antibody response was defined as anti-HBs titre ≥10 IU/L. Based on this definition, 52 HIV cases were responders to HBV vaccine and 40 cases were non-responders.

The mean age of responders and non-responders was 38.6 ± 9.3 and 37.1 ± 6.1 years, respectively; 51.9% of responders were men and 48.1% were women. In non-responders, 57.5% of cases were men and 42.5% were women. The mean CD4 count of responders and non-responders was 504 ± 234 and 320 ± 166 cells/mm³, respectively; 27.5% of responders and 40% of non-responders were anti-HCV positive. The mean ALT levels in responders and non-responders were 38.3 ± 18.8 and 39.6 ± 19.3 IU/l, respectively. There was no significant difference between responders and non-responders regarding age, sex, CD4 count and HCV infection.

HBV-DNA presence was evaluated in all cases within 1 year after HBV vaccination completion. HBV-DNA was detected in two (2.17%) of the cases. One of the OBI cases was in the responder group (HBV-DNA level: 120 IU/mL) and the other one was in the non-responder group (HBV-DNA level: 100 IU/mL). Both of the OBI subjects were women and anti-HCV negative.

Glycine to arginine mutation at residue 145 (G145R) within the ‘a’ region of the S gene was detected in one of the OBI cases who was in the non-responder group (IR 1 in Figure 1). The other OBI subject showed no mutation in the S gene (Figure 1).

Figure 1.

Position of amino acid substitutions in occult HBV isolates. The isolates found in this study are indicated as IR 1 and IR 2.

Discussion

HBV/HIV co-infection is common, due to similar routes of transmission.¹ In HBV/HIV co-infected cases, HIV can cause higher rates of HBV chronicity, decreased rates of anti-HBs seroconversion and increased viral replication. As a consequence, HBV/HIV co-infection is associated with increased liver fibrosis progression and increased rate of liver failure, cirrhosis and hepatocellular carcinoma.⁵ So HBV vaccination is highly recommended for non-immune HIV-positive patients.²⁴

Since the 1990s, HBV vaccine escape mutants have been shown to cause infection in vaccinated individuals^25,26 and vaccination might have increased selection pressure on the emergence of these mutants in relation to wild-type HBV.²² The vaccine escape mutants were mainly detected as amino acid substitutions in the ‘a’ determinant region of the S protein (residues 124–147 in the S domain), the main target of neutralising antibodies following vaccination. The most common mutation is glycine to arginine substitution at residue 145 (G145R). The mutations decrease the binding of antibodies against wild-type S protein to virions and subviral particles, which causes breakthrough infections and diagnostic failure.^16,27,28

A study in Taiwan demonstrated an increase in the prevalence of S gene mutants in infants from 7.8% before universal HBV vaccination to 23.1% after 15 years of universal vaccination against HBV.²⁹ Nainan et al.³⁰ reported that 51% of studied infants were infected with one or more S variants. They showed that the prevalence of these mutants is much higher than previous reports and emphasised the need for monitoring of HBV variants in the immunised world. Breakthrough infections with S gene mutants were also reported in Gambia, Alaska and Taiwan.^11,31,32

Mutants play a crucial role in the natural history of hepatitis B and may be important in hepatocarcinogenesis or development of fulminant hepatitis.³³ Transmission of these escape mutants could imperil HBV vaccine efficacy and has public health implications.^25,26 Besides, S mutations induce a structural change within the ‘a’ determinant region, which causes significant changes within the antigenic epitope of HBsAg and have a possible role in the pathogenesis of OBI.³⁴ Therefore, emergence of these mutants due to the global effect of vaccination programmes can lead to OBI and trying to detect this covert condition could be helpful for defining better preventive and therapeutic strategies.³⁴

To our knowledge this is the first study on the presence of OBI and S gene escape mutants in HIV-positive patients after HBV vaccination. In our study, OBI was detected in two cases: one in a responder and one in a non-responder. Glycine to arginine mutation at residue 145 (G145R) within the ‘a’ region of the S gene was detected in one of the OBI cases who was in the non-responder group. The presence of an escape mutant in our non-responder case may be explained by lack of immune attack in vaccine non-responders. In fact, HBV vaccine is a T-cell dependent antigen; other aspects of immune function including antigen presentation of the peptide-based vaccine as well as B cell activity are also important in positive serologic response to HBV vaccine. The functions of T and B cells in HBV vaccine non-responders are complicated. In these cases, mutants may change host susceptibility or may lead to escape from immune attack.³⁵

Our study showed that the prevalence of OBI and vaccine escape mutants was low in our HBV-vaccinated HIV patients in both responder and non-responder groups. HBV prevalence has decreased dramatically in the Iranian population during the last decade, and now Iran is classified as a low endemicity area for HBV. In 1993, the HBV vaccine was included in the Expanded Program on Immunization (EPI) throughout the country. After 13 years of HBV vaccination, its coverage has reached an appropriate level from 62% in 1993 to 94% in 2005.³⁶ Free HBV vaccination for HIV patients was started in Iran in 2003 and its immunogenicity varies from 29.1% to 56.6% in different studies.^37–40 Therefore, the low rate of OBI in this study could be expected due to the decreased rate of HBV in the general population and in HIV patients.

In conclusion, our study showed that the prevalence of OBI was low in our HBV-vaccinated HIV patients in both responder and non-responder groups. Besides, the selection of vaccine escape mutants following HBV vaccination was uncommon in our HIV patients. So there was no alarming evidence indicating breakthrough HBV infection in our vaccinated HIV cases. Since the prevalence of the S gene mutation is quite different in various countries, the observation might be region restricted. Therefore, a similar study is needed for other countries in order to determine the universal risk of breakthrough HBV infection in vaccinated HIV-positive patients.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors are grateful to Pasteur Institute of Iran for financial support of this study.

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