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Immune response to hepatitis B vaccine: An evaluation

Background: Hepatitis B virus infection is a major public health problem in India, and all health-care workers (HCWs) need to be immunized to prevent occupational exposure. This study was done to find the hepatitis B vaccination rates, immune response, and predictors of titer <10 mIU/ml among students and HCWs of a tertiary care institution in the state of Kerala. Materials and Methods: This was a cross-sectional study conducted in the Department of Microbiology, Government TD Medical College, Alappuzha, Kerala, India, for a period of 1½ years between January 1, 2016, and June 30, 2017. Vaccination rates were collected through a screening proforma. Of the 1321 participants who filled the screening proforma, 5 ml of blood was collected aseptically from 579 participants who were fully vaccinated (all the three doses of vaccine) and stored at −20°C until antibody to hepatitis B surface antigen (anti-HBs) assay was done using Microlisa (Biorad). Data were analyzed using SPSS for Windows, version 16.0. (SPSS Inc., Chicago, IL, USA) (trial version). Statistical Analysis: Descriptive data were expressed using frequencies and percentages, and Chi-square test was applied to find the association between antibody titer <10 mIU/ml and independent variables. Results: Of the 1321 participants who filled up the screening proforma, the vaccination rate was 72.6%. Majority of the participants, i.e. 83.5% of doctors, 81.1% of nurses, 69.7% of students, and 21.4% of technicians, had taken all the three doses of hepatitis B vaccine. Of the fully vaccinated (n = 959) participants, 76.9% had the vaccination during adulthood and only 26.1% had it during childhood. The correlate of protection was defined as the presence of anti-HBs ≥10 mIU/ml. The mean antibody titer was 448 ± 284.97 mIU/ml ranging from 9.8 to 2000. Of the 579 participants whose titer was checked, 71 (12.3%) had a nonprotective titer of <10 mIU/ml. We found that age >35 (odds ratio [OR]: 3.85, 95% confidence interval [CI]: 2.12–6.99]), last dose >10 years (OR: 5.01, 95% CI: 2.94–8.55), no boosters or revaccination (OR: 2.94, 95% CI: 1.42–6.07), and body mass index (BMI) >25 (OR: 2.51, 95% CI: 1.44–3.39) were associated with nonprotective titer. Conclusion: More than a quarter of the study population who were at high risk of exposure were unvaccinated or partially vaccinated. Even after taking the full course of hepatitis B vaccine, 12.3% had titer <10 mIU/ml. Nonprotective titer was found to be associated with age >35 years, last dose taken >10 years ago, no boosters/revaccination, and BMI ≥25.


Hepatitis B virus (HBV) infection causes acute and chronic
hepatitis through horizontal and vertical transmission and accounts for disease burden of 50 million cases in India with a prevalence of 2%-7%, amounting to an intermediate endemicity. [1] Hospital-based studies conducted in Kerala reveal a prevalence of 6.3%. [2] While acute hepatitis B characterized by acute inflammation and hepatocellular necrosis is self-limiting, chronic hepatitis B with persistence of hepatitis B surface antigen (HBsAg) in blood and serum for more than 6 months presents in myriad forms ranging from carrier state with no clinical manifestations to cirrhosis, end-stage liver disease, or hepatocellular carcinoma. [3,4] Long-term viral suppression, which remains the only modality of treatment, is challenging owing to the poor adherence due to high cost, causing a vicious cycle of disease progression and transmission. Vaccination strategies and health education need to be aggressively imparted in high-risk population which also includes the health-care workers (HCWs).
Hepatitis B vaccination for HCWs is a key component of the WHO Hepatitis B Elimination Strategy 2016-2021. [5] In developing countries, 40%-65% of HBV infections among HCWs are attributable to percutaneous occupational exposure, whereas the corresponding risk in developed countries is as low as 10%. [6] HBV is more infectious than human immunodeficiency virus or hepatitis C virus and as little as 10 −5 mL of blood can be infectious. [7] HBV vaccine has been available since 1982 and recombinant DNA vaccine since 1987 with a seroprotection of 85%-90%. [8] HBV vaccination has been adopted in the Universal Immunization Programme in India in 2007-2008 following its recommendation by the World Health Organization in the 1990s, and several studies have been done on vaccination efficacy among children. [3,9] Occupational Safety and Health Act recommends HBV vaccine followed by the confirmation of vaccine response in all HCWs. [10] However, despite the high risk of contracting the disease among HCWs and medical professional students, the vaccination is only voluntary. Even though admissions to medical professional colleges in Kerala mandate hepatitis B vaccination, the vaccination status is not followed up, leading to incomplete vaccination, amounting to an unprotected status. This study was conducted with objectives to assess the hepatitis B vaccination rates in the study population, to evaluate the immune response to hepatitis B vaccine in students and HCWs, to determine the duration of persistence of antibody to hepatitis B surface antigen (anti-HBs) after vaccination, and to find the association of titer <10 mIU/ml by enzyme linked immunosorbent assay (ELISA) method with independent variables.

MATERIALS AND METHODS
This was a cross-sectional study conducted in the Department of Microbiology at Government TD Medical College, Alappuzha, for a period of 1½ years (January 1, 2016-June 30, 2017) after getting clearance from the Ethics Committee (EC) (EC/02/2016 dated December 26, 2015, registered with the Central Drugs Standard Control Organisation).The participants were requested to fill the proforma after obtaining the informed consent from them. Inclusion criteria for titer estimation included completion of full course of hepatitis B vaccine (0, 1, and 6 months). Exclusion criteria included unvaccinated and partially vaccinated individuals, those with a past history of hepatitis B infection, or those on prolonged steroid therapy. A sample size of 567 was calculated based on the failure of seroconversion to hepatitis B vaccine in previous studies. [8] Five milliliters of blood was collected aseptically from each participant and stored at −20°C until anti-HBs assay was done using quantitative ELISA (direct antibody sandwich enzyme immunoassay using Microlisa, BIO-RAD Laboratories, Redmond,WA 98052, USA). For each assay, anti-HB-negative control and calibrators corresponding to 10, 100, 400, and 1000 mIU/ml of anti-HBs were used. The correlate of protection (CoP) was defined as the presence of anti-HBs ≥10 mIU/ml. [11] For quantitative interpretation, a graph was plotted with the optical density of controls against their assigned concentrations, using a linear regression with Microsoft Excel 2010 and equation formulated. The participants having inadequate titer values <10 mIU/ml were tested for HBsAg, and HBsAg-negative individuals were advised to take three doses of hepatitis B vaccine and report after 6 weeks after the third dose for recheking the anti-HBs titre as shown in Figure 1. The data were analyzed using the Statistical Package for the Social Science SPSS for Windows, version 16.0. (SPSS Inc., Chicago, IL, USA) (trial version). Descriptive data were expressed using frequencies and percentages, and Chi-square test was applied to find the association between antibody titer <10 mIU/ml with variables such as age >35 years, female gender, body mass index (BMI) ≥25, smoking status, alcoholism, booster dose status, years elapsed after the last dose, and presence of chronic disease.

RESULTS
A total of 1321 participants with a mean age of 24.98 ± 7.9 years filled up the screening proforma, of which 965 (73.1%) were female and 356 (26.9%) were male. The vaccination rate was 72.6% as 959 participants were fully vaccinated with 0, 1, and 6 doses of hepatitis B vaccine as shown in Figure 2, and they were invited for antibody titer estimation. Around 312 (23.6%) were partially vaccinated with 0 or 0 and 1 doses and 50 (3.8%) had not taken any dose of hepatitis B vaccine. Of the 50 participants who were not vaccinated, 15 (30%) were nurses, 16 (32%) were students, 18 (36%) were technicians, and 1 (2%) was a doctor. The vaccination rate was 60.7% among males and 70% among females. Of the 823 students screened, 614 were MBBS, 177 were BSc Nursing, and 32 were BDS students. Among them, 371 (45.08%) MBBS, 170 (20.65%) BSc Nursing, and 32 (3.8%) BDS students had taken primary series of vaccination. Of the 959 participants who were fully vaccinated, 222 (23.1%) had vaccination during childhood and 737 (76.9%) during adulthood. Only 21 (0.4%) participants reported developing adverse event following immunization in the form of pain at the site of injection, rashes, dizziness, and weakness of the whole body after primary vaccination in the screening proforma.
Five hundred and seventy-nine participants who completed primary vaccination with three dose series of hepatitis B vaccine were recruited for anti-HB assay. Their mean age was 25.5 ± 7.65 years, and the majority of participants fell in the 18-30 years' age group (81.5%) followed by 31 (124) were taken by participants who had childhood vaccination, of which 113 had taken revaccination, three had taken two booster doses, and the rest single dose of the booster.
The mean anti-HB antibody titer was 448.3 ± 284.97 mIU/ml ranging from 9.8 to 2000. Of the 579 participants, 71 (12.3%) had titer <10 mIU/ml, 36 (6.2%) had titer at 10-100 mIU/ml, 465 (80.3%) had titer at 100-1000 mIU/ml, and 7 (1.2%) had titer between 1000 and 2000 mIU/ml. Of the 71 participants with nonprotective titer, 9 had taken a single booster (8 after childhood and 1 after adult vaccination). All the 71 participants with nonprotective titer were advised revaccination with hepatitis B vaccine. Eight of them reported back after revaccination with 3 doses, after 1½ months of the third dose and had protective titers. We did not identify any nonresponder (who has titer <10 mIU/ml after revaccination with secondary three-dose series of hepatitis B vaccine).
On comparing the association of year of vaccination with titer, it was seen that out of the 71 participants with titer <10 mIU/ml, 31 (43.7%) had last vaccination more than 10 years ago (Chi-square 40.28, P < 0.001), as shown in Figure 3. There was a weak negative correlation (Pearson's R −0.259, P < 0.001) with antibody titer and increasing years of vaccination, as shown in Figure 4. An antibody titer of 350 mIU/ml was seen in a participant who had taken the vaccine 25 years back.
As summarized in Table 1, the nonprotective titer <10 mIU/ml was found to be associated with age >35 years, last dose taken >10 years ago, no boosters/ revaccination, and BMI ≥25. Gender, smoking, alcoholism, and chronic illness such as diabetes mellitus were not associated with titer <10 mIU/ml.

DISCUSSION
The occupational risk associated with hepatitis B among health-care professionals has been identified long back. Vaccine-induced seroprotection is a useful surrogate of its efficacy, and we estimated the anti-HBs antibody titers to check the protection status of the health-care students and professionals at our institution. The nonresponders of vaccine are unprotected and are at a threat to develop the disease after accidental exposure to hepatitis B. In HCWs, early identification of nonresponders is a must by checking the titer levels so that they adopt better personal protective measures rather than being in a state of false sense of security. [8,12] The Centers for Disease Control and Prevention (CDC) guidelines recommend anti-HBs titer measurement after 1-2 months of the last vaccine. [13] A health-care professional is considered immune to hepatitis B only if the person has completed the three doses of vaccine and has anti-HB titer >10 mIU/ml. [13] Of the 1321 health-care professionals screened, 959 who had completed single series of vaccination with or without booster doses were eligible to be enrolled for titer assay. The percentage of HCWs who satisfied the inclusion criteria and considered immunized were 83.5% of doctors, 81.1% of nurses, 69.7% of students, and 21.4% of laboratory technicians. Studies have shown variable vaccination rates from 33% to 83% among HCWs in various states in India. [14][15][16] Abidi et al. in a study found that the overall vaccination rate was higher among doctors than nurses. [17] In the present study, higher vaccination rate was seen in females as 70% of them were vaccinated compared to 60.7% of males. In the study by Patil et al., the vaccination rate among the doctors was lowest as only 72% of them were fully vaccinated as compared to 93% of nurses and 82% of other staff. [16] Studies show that vaccine is effective in 90%-95% of adults by protecting against HBV infection. [18] Globally, immune response following primary vaccination against hepatitis B has been found to be 55%-100%. [8] In this study, titers of 579 participants were checked after primary vaccination, of which 161 (27.8%) had taken boosters/revaccination. The immune response was 87.7% as compared to 96.4% by Purvi et al. and 97.5% by Kollathodi et al. [8,19] Of the 71 (12.3%) participants who had titer <10 mIU/ml, only 8 reported back after revaccination and had 100% seroconversion. Hence, we could not identify any nonresponders. Studies elsewhere have shown seroconversion of 89.24%-100% after revaccination. [8,19,20] Booster doses are dose of hepatitis B vaccine administered after primary vaccination series to provide rapid protective immunity against significant infection. However, there is no point in taking booster doses without checking the anti-HBs titer as the responders to boosters vary by population and age at receiving the primary hepatitis B vaccine. [13] In 2016, the World Health Organization has done away with the concept of booster doses. [5] It is recommended that in those with anti-HBs  titer <10 mIU/ml after primary vaccination, a secondary vaccine series of three doses has to be given. Those with titer >10 mIU/ml following revaccination are protected for life. [5] I n o u r s t u d y, we f o u n d t h a t n o n p r o t e c t ive titer <10 mIU/ml was found to be associated with age >35 years, last dose taken >10 years ago, no boosters/revaccination, and BMI ≥25. We could not associate gender, smoking, alcoholism, and chronic illnesses such as diabetes mellitus with titer <10 mIU/ml. Several studies have shown that there is an inverse relation of age with vaccine immunogenicity and as found in our study, age more than 35 years had 3.85 times chance to develop poor response to vaccine. [19,21] This is in contrast to that reported by Purvi et al., in whom patients of all age groups under the study had a seroconversion of 95%-97%. [8] In tune with the inverse relation of immunogenicity with age goes the statement of decline in immunogenicity of vaccine as years run by after vaccination. We could establish a weak negative correlation of years after vaccination with that of the anti-HBs antibody titer, which is similar to the results observed elsewhere. [8,19,22] When time since vaccination was considered, the protection rate declined and there is statistically significant reduction in anti-HBs titers beyond 10 years of postvaccination. Despite this, we could find a titer of 350 mIU/ml in a participant who had taken the last dose of vaccine 25 years back. This is in line with Gilca et al. who reported a persistent protective level of anti-HBs antibodies after 22 years of primary doses. [23] We found that the risk for having titer <10 mIU/ml was 2.9 times higher in those who were immunized with primary vaccination alone without a booster. However, this may be viewed skeptically as the current recommendation is to take a second series of vaccination if the titer is <10 mIU/ml and blanket boosters are no longer advocated. In this study, majority of the participants had taken booster doses or revaccination without checking the titer after primary vaccination. The CDC recommends booster vaccine only for hemodialysis patients and immunocompromised patients with HIV, hematopoietic stem-cell transplant recipients, and patients receiving chemotherapy. [24] For nonresponders, the current recommendation is a full-course revaccination. [5] A vaccination course is said to be complete only if three doses of the vaccine have been taken and the antibody titer is checked and documented. This has to be made a mandatory requirement for all health-care professionals. In government medical colleges in Kerala, it is recommended to take hepatitis B vaccine at the time of admission to medical, dental, and paramedical courses. Yet, it is a sad fact that there is no proper follow-up of the actual protection status on whether the student has completed the course of vaccine and developed protective titers. Instead of asking vaccination certificate against hepatitis B, the anti-HBs titer assay should be made mandatory for students being enrolled as health-care professionals to prevent inadvertent revaccination among those students who had taken childhood vaccination.
We found that overweight participants with BMI >25 had a higher risk of developing nonprotective titer. This is in concurrence with several studies published so far which stated that obesity was a predictor of poor response to the vaccine. [3,[25][26][27][28] This may be attributed to the effect of deposition of HBV vaccine in adipose tissue rather than in muscle, leading to the denaturation of antigen by enzymatic action and hormonal effects in overweight individuals. [3,[25][26][27] Yang et al. stated that in overweight persons, there can be damaged proliferation and function of the antibody-secreting plasma cells. [3] Liu et al. stated that the mechanism responsible for decreased immunization responses in obesity included leptin-induced systemic and B cell intrinsic inflammation, impaired T cell responses, and lymphocyte division and proliferation. [29] However, recent studies done by Purvi et al. and Kollathodi et al. did not find any association of BMI with that of nonresponse to hepatitis B vaccine. [8,19] We could not find any relation of gender with nonprotective titers, which is comparable to those published elsewhere. [8,19,25] There are contrasting results regarding the association of gender with titer <10 mIU/ml as some studies show that it is males [3] who have decreased response, while others show that it is females. [10,26] Yang et al. stated that only few immunological genes are expressed on the Y chromosome unlike the X chromosome and testosterone damages the production of immunoglobulin (Ig) IgG and IgM from B-lymphocytes and restrains the production of interleukin-6 from monocytes as opposed to estrogen which activates them, which influences the vaccine response. [30] Although some studies have shown that smoking and chronic diseases result in negative response with no-protective titers, we could not establish any association. [3,6,19,25] In line with Kollathodi et al., we could not establish an association of alcoholism with anti-HB titer <10 mIU/ml. [19] This may be attributed to the participation of low number of participants with smoking, alcoholism, and chronic disease as in the case of Purvi et al. [8] Alcohol due to its ability to cause cirrhosis has been incriminated to be an independent risk factor of poor immune response. Literature shows that nicotine and persistent alcohol can alter immune response by affecting the antigen-mediated T cell pathway and intracellular calcium response. [3] Antigenic subtypes of HBV exist, but immunization provides immunity to all subtypes because of the presence of a common antigen. When immune memory cells are present, the anti-HBs responses are immediate within 7-10 days following an exposure, and those with titer >10 mIU/ml 1 month after a dose of vaccine may be a primary immune response or it can be an anamnestic response. [31] T helper cell responses that mediate the memory of B cells have been induced with a titer >10 mIU/ml. Anamnestic response that prevents disease and infection is initiated when a vaccinated person is exposed to HBV. [11] Hence, we have to revaccinate persons who have titer <10 mIU/ml following primary vaccination. We found that all the eight patients who had revaccination had seroconverted with good titers. Sixty-three out of the 71 participants (88.7%) who had titer <10 mIU/ml and advised revaccination did not report back for titer estimation. This shows the lack of awareness, lackadaisical attitude, or busy professional commitment, which requires intervention. Following this, the Hospital Infection Control Committee of our institute drafted policies for compulsory immunization followed by titer checking in new recruits of HCWs.

Limitations
Majority of the persons who had titer <10 mIU/ml who were advised revaccination did not report back for titer estimation. We could not get proper data on the brand of vaccine used. We could not get any authentic data on the vaccination status of the participants through health cards or previous antibody titer reports and had to believe what the participants had written in the screening proforma.

CONCLUSION
More than a quarter (362) were unvaccinated or partially vaccinated among the screened participants (1321), and they were at high risk of exposure. Even after taking a full course of hepatitis B vaccine, 12.3% had lower than CoP (<10 mIU/ml). Nonprotective titer was found to be associated with age >35 years, last dose taken >10 years ago, no boosters/revaccination, and BMI ≥25. We could not find any association of gender, smoking, alcoholism, and chronic illness with nonprotective titer in this study. This study brings forth the need for initiating awareness program regarding the need for immunization against HBV in health-care professionals and students. Vaccine should be made available free of cost for all HCWs and students and they should be followed up by checking and documenting anti-HBs antibody titer. Records should be maintained regarding their hepatitis B vaccination status.