The study was conducted in Pediatric ART Clinic of a tertiary hospital in Delhi between November, 2017 and April, 2019. Approval was obtained from the institutional ethics committee and the trial was registered with the Clinical Trials Registry of India. Permission was also obtained from Delhi State AIDS Control Society.

HIV-infected children aged 18 months to 12 years who had been receiving ART for at least 6 months, were previously unimmunized for hepatitis B and were seronegative for Hepatitis B virus (HBs antigen negative) were included. The immunization status was ascertained on the basis of previous immunization records and a negative anti-HBs status. Any child with immunological failure, as defined by National AIDS Control Organization (NACO), was excluded [19].

A written informed consent was taken from the parent or guardian. Participants were randomized by computer generated software using block randomization technique with variable block sizes. Allocation to 3-dose and 4-dose groups of the study was done using concealed envelope technique. Recombinant HBV (rHBV) vaccine (Biological E. Limited) was administered to the participants in the immunization clinic while observing all universal precautions. Children in group 1 received 20 µg of rHBV vaccine IM (in deltoid muscle) at 0, 1, and 6 months, and those in group 2 received 20 µg of rHBV vaccine IM at 0, 1, 2 and 6 months. Any adverse event following immunization was reported to the appropriate authorities.

Two mL venous samples were drawn for estimation of anti-HBs titers at beginning of the seventh and twelfth month after receiving the first dose. Additionally, in the group receiving 4-dose vaccination, a sample was also drawn one month after the third dose to be assessed as an accelerated 3-dose schedule (0,1 and 2 months). The sera were separated and stored at -20 ºC.

Fifty participants were recruited in the study between November, 2017 and April, 2018. Fig. 1 depicts the flow of participants in the study. The baseline characteristics of participants in 3-dose and 4-dose groups were comparable (Table I).

Fig. 1 Flowchart showing the recruitment of participants in the study.

The median anti-HBs titers and seroprotection rates achieved in the seventh and twelfth month in both groups are shown in Table II. No serious adverse event following immunization (AEFI) was reported in any child in either group.

The proportion of responders one month after completion in accelerated schedule were 92% which was statistically comparable to the corresponding figures in the 3-dose schedule (80%; P=0.42) and 4-dose schedule (96%; P=0.08). The median anti-HBs titers in the accelerated schedule were 179.9 (130.6-250) IU/L, which were comparable to the 3-dose schedule (P=0.26), but significantly lower than the 4-dose group (P=0.03) at the seventh month.

The study concluded significantly higher median anti-HBs titers in the 4-dose group as compared to 3-dose group at seventh month, but not at twelfth month. The proportion of responders and good responders in both groups were comparable at both time points. This emphasizes that offering a fourth dose of recombinant hepatitis B vaccine in HIV-positive children on ART may be unnecessary.

We found seroprotection rates of 96% and 80% one month after HBV vaccination in the 4-dose and 3-dose schedule, respectively, similar to an adult study [9]. Seroconversion rate of 94% was likewise seen after a 4-dose (double dose) schedule in unimmunized HIV-infected Indian children [21]. Similarly, a higher serocon-version rate of 95.4% was seen in seventh, than 88.6% in the twelfth month after double dose 4-dose vaccination in adults [22]. Seroconversion rate after 3-dose double dose vaccination in HIV infected children and adults vary from 60-74% [12,23,24]. The seroconversion rates at both time points in our study were either comparable or higher than the aforementioned studies. This may be accounted for by regular ART intake for atleast six months in the present study group. Universal ART in children leading to immune reconstitution and improved vaccine response appears to be a possible explanation for the robust seroconversion rates of participants in our study.

The results of our study indicate that a 3-dose schedule may be adequate for primary immunization of these children with the added advantage of having better compliance and better use of resources, while ensuring effective protection against hepatitis B. Antenatal care and prevention of parent to child transmission (PPTCT) services have improved nationwide, which have led to more timely diagnosis in mothers and children. Further, all HIV-infected children are now routinely receiving ART irrespective of clinical and immunological staging. Well-equipped ART clinics with trained doctors and paramedical staff help in better follow-up, medical care and awareness among these patients. This has led to the improved immunological status of HIV positive children and subsequently more effective response to immunization.

The 4-dose schedule had higher median value of anti-HBs titers one month after completion of the vaccination schedule, than the 3-dose schedule. This difference was not sustained as the titers continued to rise in the 3-dose group. However, whether the greater proportion of good responders in the 4-dose group compared to the 3-dose group will offer longer duration of seroprotection, needs to be confirmed with a longer follow up.

The seroconversion rate increased between seventh to twelfth month in the 3-dose group but remained static in the 4-dose group in the present study, similar to that reported before [25]. The increase in the proportion of seroconverters over time in our study, however, implied that those who do not seroconvert initially may show gradual increment in titers over time. This may be explained by gradual immune reconstitution with continued ART in HIV-infected children leading to a delayed immune response to vaccination.

The timely immunization and sampling were ensured in the present study without any lost to follow-up. Earlier studies in children and adults were conducted when ART was not being administered universally, unlike the present study where all participants universally received HAART. Limitation of our study is the small sample size. Implementation of universal national immunization schedule and practices makes it difficult to establish a big cohort of unimmunized HIV positive children. However, the fact that nearly 11% of children were found to be unimmunized, emphasizes the need to strengthen the immunization services for this vulnerable group. Further, due to the non-availability of viral load and tests for B-cell and T-cell functions, the non-response to vaccination could not be explained in a few participants who did not qualify for severe immunosuppression based on their CD4 counts.

We suggest that three doses of double strength hepatitis B vaccine may suffice in HIV-infected children receiving ART in the absence of immunological failure. The accelerated 3-dose schedule (0, 1, 2 months) may also be studied for its long term immunogenicity before it can be considered as an alternative regimen for vaccination of these children.

Ethics clearance: Institutional Ethics Committee, UCMS; No. IECHR/2017/32/100 dated 17 October, 2017.

Contributors: PD, PJ, SG, BK, AR: conceptualized the study; PJ, PD: data collection; BK: laboratory support; PD, PJ: drafted the manuscript; SG, BK, AR: critical input. All authors approved the final manuscript and are accountable for the manuscript.

Funding: Intramural grant, University College of Medical Sciences.

Competing interests: None stated.