Effects of Risk Factors on Anti-HBs Development in Hepatitis B Vaccinated and Nonvaccinated Populations

Abstract

Hepatitis B infection is still a major global health problem even though safe and effective vaccines have been available for more than 30 years. Although development of protective antibody to hepatitis B surface antigen (anti-HBs) is a common phenomenon after vaccination as well as natural infection, sometimes it does not appear even after complete vaccination. In the present study, whether the impairment of the development of anti-HBs in naturally infected and/or vaccinated populations is associated with immunomodulating risk factors (i.e., age, gender, smoking, and diabetes) and/or other risk factors (i.e., socioeconomic status, dental, and saloon exposure) was investigated through a cross-sectional study. Among 204 nonvaccinated patients, 132 (64.7%) tested positive for anti-HBc, indicating that they had been exposed to hepatitis B virus (HBV) at least once in their lifetime. Exposure to HBV (anti-HBc positive) was significantly higher among low-income people, dental exposed, and saloon users. Among anti-HBc positive patients, only 44 (33.3%) developed natural immunity with anti-HBs. Impairment in anti-HBs formation was found to be significantly high among cigarette smokers. However, no significant association of anti-HBs development was observed with age, gender, socioeconomic status, diabetes, dental exposure, and using saloon. Consistently, the frequency of developing protective anti-HBs (≥10 IU/L) among a vaccinated population was almost nine times less among smokers. These data suggest that anti-HBs development, either naturally or after vaccination, is significantly lower among smokers. It emphasizes the need to check the anti-HBs status in smokers after vaccination, and a booster vaccination should be administered if the anti-HBs antibody titer decreases below the protective level.

Introduction

Hepatitis B virus (HBV) is a major etiological agent of viral hepatitis causing significant health complications, including both acute and chronic liver diseases (16,20). Globally, more than 300 million of people are chronically infected with HBV, and approximately 1.2 million people die each year from chronic HBV infection, liver cirrhosis, and liver cancer (10,11,18). Hepatitis B is found worldwide, but it is particularly prevalent in Asia, the Middle East, and Africa (7,12,14).

The status of HBV infection in an individual is usually predicted by detection of HBV serological markers, although it is often found to be misleading (21). In general, hepatitis B surface antigen (HBsAg) is detectable during the clinical incubation period (average 2–3 months), and it disappears in convalescence (8,15,24). The prolonged presence of HBsAg (>6 months) indicates carrier state or chronic hepatitis (26). Antibody to hepatitis B core antigen (anti-HBc) develops in response to natural HBV infection that has no protection ability but appears as early as 6 weeks after infection and has lifelong persistence (3,4). In contrast, protective antibody to hepatitis B surface antigen (anti-HBs) is supposed to develop during convalescence after acute HBV infection as well as after hepatitis B vaccination but not in chronic infection (2).

However, prior studies have demonstrated that presence of anti-HBc without anti-HBs and HBsAg is frequently found in HBV serological tests (3,22), although there is only a short window when HBsAg has disappeared but anti-HBs has not yet developed that characterizes this phenomenon (25). Thus, one can anticipate that there could be other reasons that have impaired the development of anti-HBs or made anti-HBs undetectable. It is thought that anti-HBs remains undetectable in the laboratory tests because of binding to the large amount of HBsAg present in the blood as well as binding to immune complexes (19). However, the association of anti-HBs development in a HBV-exposed population (anti-HBc positive) has not yet been investigated. Anti-HBs development after vaccination has been shown to be associated with age, occupation, smoking, complete and schedule vaccination, and time of the last vaccination, but not with gender (5). Nevertheless, female vaccine recipients usually develop a higher anti-HBs level against HBsAg than males (1,9). This discrepancy suggests that the immune response may differ in people from different regions due to different life-styles, weather, food habits, and genetic factors (5).

In this study, the levels of HBsAg, anti-HBs, and anti-HBc were examined in the serum of hospital outdoor patients in Dhaka city to find out the association of anti-HBs development with different risk factors, namely, age, gender, socioeconomic status (SES), smoking, diabetes, dental exposure (visit dentists for dental treatment), and using saloons (barber shops) for shaving and haircuts in nonvaccinated and vaccinated populations in Bangladesh.

Materials and Methods

Sample collection

The serum samples were collected during April–November 2013from 204 nonvaccinated hospital outdoor patients from Dhaka city with nonspecific symptoms after obtaining informed written consent. The study protocol was approved by the ethical committee of Dhaka Medical College (reference number: DMC-MEU/ECC/2014/16). All of the participants were asked to complete a questionnaire that included information about their age, gender, SES, smoking habits, diabetes status, dental exposure, and use of saloons. Maintaining proper aseptic condition, approximately 5 mL of fresh blood was drawn from each participant. The blood was then allowed to clot for approximately 10 min before being centrifuged at 2,000 g for 5 min to separate the serum. Serum samples were kept at −80°C until use.

Secondary data comprising the anti-HBs titers of 56 vaccinated healthcare workers (HCW) from East West Medical College Hospitals, Dhaka, Bangladesh, were collected after obtaining their informed written consent. These HCWs were vaccinated as per their job recommendation, and their immune response was examined after three doses of the complete vaccination program.

Serological tests

Serum samples of nonvaccinated hospital outdoor patients were analyzed for detection of HBsAg, anti-HBs, and anti-HBc by enzyme-linked immunosorbent assay (ELISA) using kits (JAJ International, Inc.) as per the manufacturer's instructions. The anti-HBs titer in the serum of vaccinated HCW was analyzed by an automated chemiluminescent immunoassay system using kits (Siemens) according to the manufacturer's instructions.

Statistical analysis

Data were analyzed with MedCalc v11.3.0.0; the association of anti-HBs status with immunomodulating risk factors was analyzed by estimating prevalence odds ratios (POR). Statistical significance was assessed if 95% confidence interval (CI) did not include the value 1. The chi-square test was performed to calculate p-values.

Results

Presence of HBV seromarkers in relation to risk factors

Among 204 nonvaccinated hospital outdoor patients, 132 (64.7%) tested positive for anti-HBc, which suggested that if there was no false-positive result, then these positive patients had been exposed to HBV at least once in their lifetime (Table 1). Exposure to HBV (i.e., anti-HBc positive) was significantly higher among low-income patients (POR 2.4314 [95% CI 1.3441–4.3985]; p=0.005), dental exposure (POR 2.4588 [95% CI 1.2916–4.6807]; p=0.009), and saloon users (POR 2.583 [95% CI 1.1976–5.571]; p=0.02), while it was higher among males, those older than 30 years of age, cigarette smokers, and diabetes patients, but this difference was not statistically significant (Table 2).

Table 1.

Prevalence of HBV Seromarkers in Nonvaccinated Outdoor Patients

Serological profiling
HBsAg	Anti-HBs	Anti-HBc	No. of samples (%)	Interpretation
−	−	−	72 (35.3)	Susceptible to HBV infection
+	−	+	9 (4.4)	Acutely or chronically infected
−	+	+	44 (21.6)	Immune due to natural infection
−	−	+	79 (38.7)	Interpretation is unclear, four possibilities:
				I. Resolved infection (most common)
				II. False positive anti-HBc, thus susceptible
				III. Low-level chronic infection
				IV. Resolving acute infection

−, not detected; +, detected.

HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; anti-HBs, antibody to hepatitis B surface antigen.

Table 2.

Exposure to HBV Among Nonvaccinated Patients According to Socio-Demographic Characteristics

Factors	Anti-HBc positive	Anti-HBc Negative	POR (95% CI)	p-Value
Age (years)
≤30 (114)	72	42	0.8571 (0.4797–1.5316)	0.71
>30 (90)	60	30
Gender
Male (125)	84	41	1.3232 (0.7364–2.3774)	0.43
Female (79)	48	31
Smoking (only male)
Yes (56)	40	16	1.4205 (0.6645–3.0364)	0.47
No (69)	44	25
Diabetes
Yes (51)	38	13	1.8347 (0.903–3.7278)	0.13
No (153)	94	59
Socioeconomic status
Low income (127)	92	35	2.4314 (1.3441–4.3985)^*	0.005^*
High income (77)	40	37
Dental
Exposure (74)	57	17	2.4588 (1.2916–4.6807)^*	0.009^*
Nonexposed (130)	75	55
Saloon (only male)
User (77)	58	19	2.583 (1.1976–5.571)^*	0.02^*
Nonuser (48)	26	22

Low income is a total income of <US$1,200/year; and high income is a total income of ≥US$1,200/year.

Statistically significant.

POR, prevalence odds ratios; CI, confidence interval.

Association of anti-HBs detection with risk factors in nonvaccinated people

Among 132 anti-HBc positive patients, only 44 (33.3%) patients developed natural immunity with anti-HBs. Detection of anti-HBs was low among smokers (POR 0.2323 [95% CI 0.0848–0.636]; p=0.007), suggesting that the development of anti-HBs was almost five times less among smokers than nonsmokers, which was statistically significant. However, no statistically significant association was found between anti-HBs development and risk factors such as age, gender, diabetes, SES, dental exposure, and use of saloons (Table 3).

Table 3.

Development of Anti-HBs Among Nonvaccinated and Anti-HBc Positive Patients According to Socio-Demographic Characteristics

Factors	Anti-HBs positive	Anti-HBs Negative	POR (95% CI)	p-Value
Age (years)
≤30 (72)	23	49	0.8717 (0.4218–1.8014)	0.85
>30 (60)	21	39
Gender
Male (84)	28	56	1.00 (0.4713–2.1219)	0.85
Female (48)	16	32
Smoking (only male)
Yes (40)	7	33	0.2323 (0.0848–0.636)^*	0.007^*
No (44)	21	23
Diabetes
Yes (38)	10	28	0.6303 (0.2733–1.4536)	0.38
No (94)	34	60
Socioeconomic Status
Low income (92)	31	61	1.0555 (0.4788–2.3265)	0.95
High income (40)	13	27
Dental
Exposure (57)	16	41	0.6551 (0.3115–1.3777)	0.35
Nonexposed (75)	28	47
Saloon (only male)
User (58)	18	40	0.720 (0.274–1.8923)	0.68
Nonuser (26)	10	16

Statistically significant.

Association of anti-HBs detection with risk factors in vaccinated people

Observing the significant negative association of smoking with anti-HBs development in nonvaccinated patients, an attempt was made to confirm the association in the vaccinated population. The protective anti-HBs status (>10 IU/L) of vaccinated HCW was also significantly less among smokers (POR 0.1129 [95% CI 0.03146–0.4052]; p=0.001; Table 4), while the association of age, diabetes, and SES of vaccinated HCW with anti-HBs development was not significant (data not shown).

Table 4.

Unresponsiveness to Anti-HBs of Smokers in Vaccinated Healthcare Workers

Smoking	Responsive to anti-HBs	Unresponsive to anti-HBs	POR (95% CI)	p-Value
Yes (19)	7	12	0.1129	0.001^*
No (37)	31	6	(0.03146–0.4052)^*

Statistically significant.

Responsive to anti-HBs, ≥10 IU/L; unresponsive to anti-HBs, <10 IU/L.

Discussion

Similar to the observations in the current study (Table 1), numerous studies in other contexts have also found that patients expressing anti-HBc but not anti-HBs or HBsAg (i.e., anti-HBc +ve, anti-HBs −ve, HBsAg −ve) are more common than any other HBV-affected groups, such as chronically infected (anti-HBc +ve, anti-HBs −ve, HBsAg +ve) or those recovering from acute infection (anti-HBc +ve, anti-HBs +ve, HBsAg −ve) (3,22). However, the underlying reasons are poorly understood. The purpose of this study was to determine whether there is any risk factor that inhibited anti-HBs development after exposure to HBV that resulted in an increased the number of HBV-exposed people (anti-HBc +ve) developing neither chronicity (i.e., HBsAg −ve) nor immunity (i.e., anti-HBs −ve). In this regard, first anti-HBc positive patients were detected in the study population. Among them, those who expressed anti-HBs antibody were compared with those negative for anti-HBs antibody in terms of demographic characteristics and immunomodulating risk behaviors.

About 65% of the study population was found to have been exposed to HBV (i.e., anti-HBc +ve), which is relatively higher than the rate found in the general population of Bangladesh (>40%) (17). One plausible explanation is that the majority of the study population comprised low-income people, in which the HBV exposure rate is found to be significantly high (Table 2). It was also found that most of these low-income people use ordinary saloons (data not shown) where a common razor is used for shaving, which could be a plausible explanation for the high prevalence of HBV exposure among low-income people. As shown in Table 2, the HBV exposure rate (anti-HBc +ve) was significantly higher, being about 2.5 times higher among low-income people, dental exposed, and saloon users.

Among anti-HBc positive patients, anti-HBs were undetectable in about 67% of cases. One possible explanation is that anti-HBs often remains undetectable in laboratory tests because of binding to the large amount of HBsAg present in the blood as well as binding to immune complexes (19). However, in this study, it was found that impairment in anti-HBs development in anti-HBc positive people is clearly associated with cigarette smoking (Table 3), which is an immunomodulating risk factor, suggesting that these people may not have developed anti-HBs after natural HBV infection due to smoking.

For further confirmation, secondary data comprising the anti-HBs titers from a vaccinated population were analyzed, and it was found that the frequency of developing protective anti-HBs titers (≥10 IU/L) was significantly lower among smokers (POR 0.1129 [95% CI 0.03146–0.4052], p=0.001), suggesting that smokers had almost nine times less chance of developing protective anti-HBs than nonsmokers, even after complete immunization. Altogether, these data clarify that smoking is an immunomodulating factor, and it may inhibit anti-HBs production in the body. Although the mechanism by which cigarette smoking weakens the immune system is not fully understood, it is thought that cigarette smoking affects T-cell responsiveness that may account for decreased T-cell proliferation and T-dependent antibody responses in humans and animals exposed to cigarette smoke (13,23).

However, no significant association was found between anti-HBs development and age, gender, SES, diabetes, dental exposure, and the use of saloons. Previously, anti-HBs development in a vaccinated population has been shown to be associated with age and gender in other countries (5,27). A large number of samples may be required to establish these comparatively weak associations. The findings of this study suggested that occult HBV infection might also be caused, which is defined as the presence of HBV-DNA in blood or liver tissues without detectable HBsAg, with or without anti-HBc and anti-HBs (6). In Bangladesh, occult HBV infection and its association with anti-HBs development need to be demonstrated.

In conclusion, this study is unique for two reasons. First, it has investigated the risk factors associated with human immune response after natural HBV exposure, which have been poorly investigated before. Second, importantly, the degree of severity of cigarette smoking on the impairment of immunity development against HBV in both nonvaccinated and vaccinated populations has been determined, showing that cigarette smokers had almost nine times less chance of developing protective anti-HBs than nonsmokers, even after complete vaccination. Smoking prevention not only causes a reduction in non-responders (<10 IU/L) to hepatitis B vaccine but may also help the recovery and development of immunity against HBV after natural infection. This emphasizes the need to test the anti-HBs titers in smokers after complete vaccination, and a booster vaccination should be administered if the titer is below the protective level.

Footnotes

Acknowledgments

We are grateful to the University Grant Commission (UGC), Bangladesh, for their financial cooperation with this study. We also thank Md. Zaforullah Chowdhury and all the participants of this study for their cooperation.

Author Disclosure Statement

No competing financial interests exist.

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