# Abstract

 # Background The safety of the immune complexes composed of yeast-derived hepatitis B surface antigen (HBsAg) and antibodies (abbreviated as YIC) among healthy adults and chronic hepatitis B patients has been proved in phase I and phase IIa trial. A larger number of patients for study of dosage and efficacy are therefore needed. 

## Methods and Principal Findings Two hundred forty two HBeAg-positive chronic hepatitis B patients were immunized with six injections of either 30 µg YIC, 60 µg of YIC or alum adjuvant as placebo at four-week intervals under code. HBV markers and HBV DNA were monitored during immunization and 24 weeks after the completion of immunization. The primary endpoint was defined as loss of HBeAg, or presence of anti-HBe antibody or suppression of HBV DNA, while the secondary endpoint was both HBeAg seroconversion and suppression of HBV DNA. Statistical significance was not reached in primary endpoints four weeks after the end of treatment among three groups, however, at the end of follow-up, HBeAg sero-conversion rate was 21.8%(17/78) and 9% (7/78) in the 60 µg YIC and placebo groups respectively (p = 0.03), with 95% confidence intervals at 1.5% to 24.1%. Using generalized estimating equations (GEEs) model, a significant difference of group effects was found between 60 µg YIC and the placebo groups in terms of the primary endpoint. Eleven serious adverse events occurred, which were 5.1%, 3.6%, and 5.0% in the placebo, 30 µg YIC and 60 µg YIC groups respectively (p>0.05). 

## Conclusions Though statistical differences in the preset primary and secondary endpoints among the three groups were not reached, a late and promising HBeAg seroconversion effect was shown in the 60 µg YIC immunized regimen. By increasing the number of patients and injections, the therapeutic efficacy of YIC in chronic hepatitis B patients will be further evaluated. 

## Trial Registration ChiCTR.org [ChiCTR-TRC-00000022](#) # Baseline characteristics of enrolled patients Three hundred and fifty four hepatitis B patients were evaluated for the inclusion criteria. Of these, 242 patients were eligible and assigned randomly to placebo, 30 µg YIC and 60 µg YIC groups in a three-month period. Five patients were found ineligible during the batch assay at the end of treatment (week 24) at the central laboratory, and therefore were excluded from the intent-to-treat analysis. Of 237 patients included in the analysis , 8 receiving placebo, 5 receiving 30ug YIC, and 10 receiving 60ug YIC either did not complete the treatment, did not complete the follow-up or violated the protocol ( [Figure 1](#) ). No significant difference was found in baseline characteristics among three groups ( [Table 1](#) ). <figure> 10.1371/journal.pone.0002565.g001 <p><img src="" /></p> <figcaption>Summary of participants.<br /> Footnote: The numbers of SAE patients are those who discontinued treatment, while other SAE patients are not included in this figure.</figcaption> </figure> ::::table-wrap

10.1371/journal.pone.0002565.t001

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## Characteristics of participants.

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Characteristics Placebo group (N = 78) 30 µg YIC group (N = 81) 60 µg YIC group (N = 78)

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Age (yr; SD) 27.9±7.6 28.6±8.6 28.8±8.0
Female sex (no. ; %) 19 (24.4) 22 (27.2) 17 (21.8)
Weight (kg; SD) 61.7±11.1 62.3±10.0 63.7±9.3
Course of illness, hepatitis B (yr; SD) 6.3±5.2 6.0±5.8 6.1±5.4
Family history, hepatitis B (no.;%) 46 (59.0) 46 (56.8) 46 (59.0)
Alanine aminotransferase (IU/liter; SD) 169.6±80.0 162.5±74.7 171.8±93.4
HBV DNA (log copies/mL; SD) 7.1±0.9 7.1±0.9 7.2±0.8

SD: Standard deviation.:::: # Kinetics of responses in patients As shown in [Table 2](#) , a delayed response to YIC was observed between 24 weeks and the end of follow-up. For intent-to-treat analysis, at the end of follow-up, 31, 35 and 28 patients achieved the primary endpoint, and 6, 7 and 14 patients reached the secondary endpoint in the placebo, 30 µg YIC and 60 µg YIC groups, respectively. The response rate for secondary endpoint in the 60 µg YIC group was comparatively higher than that of the other groups, though statistical significance had not been reached. However, at the end of follow-up, a significant difference on HBeAg seroconversion was found between 60 µg YIC and placebo groups (p = 0.03, 2-tailed). The 95% confidence interval for this difference was 1.5% to 24.1%. In contrast, a statistical significance had not been reached between 30 µg YIC and placebo groups. ::::table-wrap
10.1371/journal.pone.0002565.t002

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## Virologic response at the end of treatment and the end of follow-up.

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```
                                      End of treatment (week 24)   End of follow-up (week 44)                                                                                                                       
```

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```
                                      Placebo group (N = 78)       30 µg YIC group (N = 81)     60 µg YIC group (N = 78)   p value   Placebo group (N = 78)   30 µg YIC group (N = 81)   60 µg YIC group (N = 78)   p value
```

HBeAg loss (No.;%) 8 (10.3) 9 (11.1) 9 (11.5) 0.99 9 (11.5) 11 (13.6) 18 (23.1) 0.13
Presence of anti-HBe antibody (No.;%) 9 (11.5) 15 (18.5) 15 (19.2) 0.65 10 (12.8) 18 (22.2) 23 (29.5) 0.06
HBeAg seroconversion [\*](#) (No.;%) 6 (7.7) 8 (9.9) 6 (7.7) 0.97 7 (9.0) 10 (12.3) 17 (21.8) 0.08
HBV DNA 13 (16.7) 21 (25.9) 21 (26.9) 0.46 28 (35.9) 21 (25.9) 29 (37.2) 0.28

> 2log decrease (No.;%)\
> Primary endpoint [ˆ;](#) (No.;%) 18 (23.1) 22 (27.2) 26 (33.3) 0.63 31 (39.7) 28 (34.6) 35 (44.9) 0.39
> Secondary endpoint [$](#) (No.;%) 3 (3.8) 8 (9.9) 6 (7.7) 0.59 6 (7.7) 7 (8.6) 14 (17.9) 0.14

: 95% confidence interval (2-tailed) for the difference of response rate
at week 44 between 60 µg YIC and placebo groups was 1.5% to 24.1%; it
was −6.3% to 13.0% between 30 µg YIC and placebo groups.

: 95% confidence interval (2-tailed) for the difference of response rate
at week 44 between 60 µg YIC and placebo groups was −10.4% to 20.6%; it
was −20.2% to 9.8% between 30 µg YIC and placebo groups.

: 95% confidence interval (2-tailed) for the difference of response rate
at week 44 between 60 µg YIC and placebo groups was −0.2% to 20.7%; it
was −7.6% to 9.5% between 30 µg YIC and placebo groups.:::: Furthermore, when applying the GEEs method to estimate the group and time effects, a significant difference of group effects was found between the 60 µg group and the placebo group in terms of the primary endpoint, and response rate calculated by both primary and secondary endpoints changed significantly over the time in the placebo, 30 µg and 60 µg YIC groups (p\<0.05) ( [Table 3](#) ). ::::table-wrap
10.1371/journal.pone.0002565.t003

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## Repeated measures analysis for time and group effects controlling by baseline ALT and HBV DNA.

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Group Primary endpoint Secondary endpoint

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```
                                 OR (95% confidence interval)   p value              OR (95% confidence interval)   p value
```

30 µg YIC group vs placebo group 0.9 (0.5--1.4) 0.52 1.3 (0.5--3.6) 0.60
60 µg YIC group vs placebo group 1.7 (1.0--2.7) 0.04 2.2 (0.9--5.4) 0.09
Week 12 vs Week 44 0.3 (0.2--0.5) \<0.0001 0.3 (0.2--0.6) 0.0006
Week 24 vs Week 44 0.5 (0.4--0.7) \<0.0001 0.6 (0.4--0.9) 0.01
Week 36 vs Week 44 0.7 (0.6--1.0) 0.04 0.6 (0.4--1.0) 0.04:::: When the baseline serum HBV DNA and levels of HBeAg from all secondary responders at the end of follow-up in the three groups were analyzed, among the 14 patients immunized with 60 µg YIC , five had HBV DNA≥10 ^7^ copies/ml, eight had HBV DNA ≥10 ^6^ and \<10 ^7^ copies/ml, only one had HBV DNA 10 ^5^ copies/ml. In contrast, of the 6 patients immunized with alum, five patients had HBV DNA 10 ^5^ copies/ml, one had HBV DNA ≥10 ^6^ and \<10 ^7^ copies/ml, none of them had HBV DNA ≥10 ^7^ copies/ml. # Reversion of HBeAg and rebound of HBV DNA The incidence of rebound in virus replication and reversion to serum HBeAg at the end of follow-up were compared among the three groups. For those responders who reached the secondary endpoint, none of the patients from either the 60 µg YIC group or placebo group showed a rebound in virus load nor in reversion to serum HBeAg. In contrast, four responders in the 30 µg YIC group showed a virus load rebound to the baseline level and HBeAg reverted to positive. Interestingly, rebound of viral load and reversion to serum HBeAg were all from responders who only reached the primary endpoint at the end of immunization, while none occurred in those who reached the secondary endpoint, suggesting that patients who achieved the secondary response at the endpoint of treatment were unlikely to develop reversion. # Adverse events and severe adverse events Overall, the most common systematic symptoms were similar for the three groups. However, more local reactions were found in the 60 µg YIC and 30 µg YIC groups. In the placebo group, the most common reaction was pain at the injection site, followed by malaise and fatigue; in the 30 µg and 60 µg YIC group, the most common adverse events were pain at the injection site, pruritus and swelling ( [Table 4](#) ). During study period, eleven patients experienced elevated ALT levels over ten times the normal level, accompanied by high levels of serum bilirubin and thus were hospitalized. ::::table-wrap
10.1371/journal.pone.0002565.t004

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## Occurrence of most common adverse events during treatment and follow-up.

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Adverse events No. of patients (%) p value

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```
                       Placebo group (N = 79)   30 µg YIC group (N = 83)   60 µg YIC group (N = 80)   
```

Local adverse reaction\
Erythema 6 (7.6) 15 (18.1) 15 (18.8) 0.08
Swelling 9 (11.4) 19 (22.9) 25 (31.3) 0.009
Pain 32 (40.5) 42 (50.6) 45 (56.3) 0.13
Pruritus 12 (15.2) 22 (26.5) 34 (42.5) 0.0006
Systematic symptom\
Fever 11 (13.9) 8 (9.6) 11 (13.8) 0.63
Malaise 16 (20.3) 12 (14.5) 12 (15.0) 0.57
Headache 8 (10.1) 9 (10.8) 17 (21.3) 0.09
Dizziness 11 (13.9) 11 (13.3) 18 (22.5) 0.24
Fatigue 14 (17.7) 19 (22.9) 19 (23.8) 0.62
Vomiting 5 (6.3) 5 (6.0) 4 (5.0) 0.94
Nausea 8 (10.1) 9 (10.8) 14 (17.5) 0.35
Abdominal pain 6 (7.6) 6 (7.2) 8 (10.0) 0.84
Diarrhoea 6 (7.6) 6 (7.2) 4 (5.0) 0.81:::: The occurrence of serious adverse events calculated according to the intent-to-treat principle was 5.1% (4/79), 3.6% (3/83), and 5.0% (4/80) in the placebo, 30 µg YIC and 60 µg YIC groups respectively (p>0.05). Ten of these were males, while one was female. The age of patients with SAE varied between 21 and 41years of age. Four occurred after the first injection (2 in placebo group, 2 in 60 µg YIC group), two occurred after the second injection (1 in placebo group, 1 in 30 µg YIC group), one occurred after 5 injections (in 30 µg YIC group), and the other four appeared after 6 injections (1 in placebo group, 1 in 30 µg YIC group and 2 in 60 µg YIC group). All 11 patients were hospitalized and recovered after treatment without immunomodulating drugs (one patient used antiviral treatment). No deaths were observed during the study period.


Extracted trial result:
Outcome: adverse events (binary)
Intervention: 30 µg YIC
Comparator: placebo
Intervention events: 3
Intervention group size: 83
Comparator events: 4
Comparator group size: 79