Recombinant HBsAg vaccine in persons with HIV: Is seroconversion sufficient for long-term protection?

Transcription

1 Recombinant HBsAg vaccine in persons with HIV: Is seroconversion sufficient for long-term protection? by Jeff Powis A thesis submitted in conformity with the requirements for the degree of Master s of Science, Clinical Epidemiology Department of Health Policy, Management and Evaluation University of Toronto Copyright by Jeff Powis 2010

2 Recombinant HBsAg vaccine in persons with HIV: Is seroconversion sufficient for long-term protection? Abstract Jeff Powis Master s of Science, Clinical Epidemiology Department of Health Policy, Management and Evaluation University of Toronto 2010 The recombinant Hepatitis B surface antigen vaccine inadequately protects those living with HIV from Hepatitis B virus infection. This study utilized saved serum samples from a retrospective cohort of persons with HIV and documented vaccine-induced HBsAb seroconversion to determine factors associated with persistence of protective levels of HBsAb ( 10mIU/ml). HBsAb levels fell below 10mIU/ml in 27% of the cohort after a median follow-up of 43 months. HIV viral load suppression (<50copies/ml) at the time of vaccination was the major factor associated with persistence of protective levels of HBsAb (OR 3.83, p <0.01). Among individuals who lost protective levels of HBsAb, booster doses of vaccine re-instated the development of protective levels of HBsAb. Delaying or repeating HBV vaccination until after suppression of HIV viral load is achieved should be considered HBV antibody levels should be followed over time and boosters given with loss of protective levels of HBsAb. ii

3 Acknowledgments This thesis would not have been possible without the support, mentoring and inspiration provided by my thesis supervisor Dr. Sharon Walmsley. My thesis committee members, Dr. Janet Raboud, Dr. Gary Naglie, Dr. David Wong and Dr. Mario Ostrowski improved this work through their valuable suggestions. Gloira Crowl, my research coordinator, worked endlessly to enroll participants for the trial and ensure the integrity of the data. Thank you to the clinics who shared patient data and laboratory staff for the retrieval and testing of stored specimens. Funding for the project was received through an operating grant provided by the Ontario HIV Treatment Network (OHTN). Dr. Walmsley and Raboud receive career support from OHTN. I dedicate this thesis to my joy and inspiration: my wife Alison, and my children Maeve and Dylan. iii

4 Table of Contents Abstract...ii Acknowledgments... iii Table of Contents... iv List of Tables... vii List of Figures... viii List of Appendices... ix Chapter 1 Introduction Rationale Study Question... 3 Chapter 2 Background Epidemiology of HBV among those living with HIV Impact of HBV among those living with HIV in the HAART era Duration of immunity and the need for boosters among those without HIV Protective immunity among individuals living with HIV... 6 Chapter 3 Methods Research Design Study Population Study Setting Study Participants Inclusion Criteria Exclusion Criteria Definitions Protective levels of HBsAb Anamnestic Response iv

5 3.3 Loss of protective immunity HIV viral load suppression HIV viral load blip Post-vaccination titer Key Predictor and Outcome variables Outcome: Loss of protective levels of HBsAb during follow-up Primary predictor variable of interest Other factors potentially associated with loss of protective levels of HBsAb Statistical Analysis Primary Analysis Secondary Analyses Sample size and power Chapter 4 Results Description of the cohort Baseline characteristics according to HIV viral load suppression at time of vaccination HIV viral load suppression at time of vaccination and loss of protective HBsAb titers Univariate analysis of factors associated with loss of protective levels of HBsAb Multivariate analysis of factors associated with persistence of protective levels of HBsAb26 4 Booster response among those with loss of protective levels of HBsAb Post-vaccination HBsAb titers as a predictor of loss of protective HBsAb titers Chapter 5 Discussion Loss of protective levels of HBsAb among those living with HIV Factors associated with persistence of protective levels of HBsAb Booster response among those with loss of protective levels of HBsAb Major study limitations Quality Improvement opportunities v

6 6 Future research directions Clinical implications and conclusions References Appendices vi

7 List of Tables TABLE 1: TABLE 2: TABLE 3: TABLE 4: TABLE 5: TABLE 6: Sample Size per group with varying assumptions for loss of protective levels of HBsAB 19 Patient demographic and clinical characteristics at time of vaccination...21 Characteristics among those with and without HIV viral load suppression at time of receipt of last dose of recombinant HBsAg vaccine..23 Univariate analysis of factors associated with loss of protective levels of HBsAb 25 Multivariable model of factors associated with loss of persistence levels of HBsAb utilizing suppressed baseline HIV viral load Multivariable model of factors associated with persistence of protective levels of HBsAb during follow-up utilizing proportion of time of followup spent with a suppressed baseline HIV viral load..27 vii

8 List of Figures FIGURE 1: Post-booster HBsAb titers levels among 19 participants with loss of protective levels of HBsAb. 28 viii

9 List of Appendices APPENDIX A: Consent Form.52 APPENDIX B: Contact Letter 59 APPENDIX C: Data Collection Form.61 ix

10 1 The purpose of this chapter is to: 1. Describe the study rationale; 2. Introduce the study question. 1 Rationale Chapter 1 Introduction Hepatitis B virus (HBV) and Human immunodeficiency virus (HIV) are common chronic viral infections with an estimated worldwide prevalence of 400 million and 40 million people respectively(1). Individuals with HIV are commonly exposed to HBV due to shared routes of transmission. In addition, after exposure the risk of developing chronic HBV infection is high compared to those without HIV (2;3). Once co-infected with HIV and HBV, there is a more rapid progression to end-stage liver disease (ESLD) and higher rates of liver-re1ated morbidity and mortality compared to individuals infected with HBV alone. In the highly active antiretroviral therapy (HAART) era, HIV-associated complications are less frequent and liverrelated deaths now account for up to 40% of deaths among persons with HIV(4;5). Treatment of HBV-HIV co-infection is challenging and limited to a small number of antiviral agents. Furthermore, until recently, patients with HIV and ESLD were not considered eligible for organ transplantation in Canada. The rapid progression of HBV-associated liver disease in co-infected individuals, the increased risk of chronic infection, and the limitations of treatment make the prevention of HBV a major public health issue among those living with HIV. There is currently available a recombinant Hepatitis B surface antigen (HBsAg) vaccine for the prevention of HBV infection. The vaccine is provided as an intramuscular injection at 0, 1 and 6 months. Production of sufficient levels of Hepatitis B surface antibody (HBsAb 10mIU/ml) in response to vaccine administration provides individuals with protection from clinically significant HBV(6-9). This process of successful HBV vaccination is termed HBsAb seroconversion. The duration of protection after successful HBV vaccination is a function of how long protective levels of HBsAb persist and, after the loss of protective levels of HBsAb, whether a significant anamnestic response is present following exposure to HBV. An anamnestic 1

11 2 response leads to the rapid production of HBsAb to above protective levels after exposure to HBV(10). Individuals with an intact immune system have seroconversion rates of greater than 95% following vaccine and have long lived protective levels of HBsAb. In addition, if protective levels of HBsAb are lost, a significant anamnestic response is present upon re-exposure preventing clinically significant HBV infection. As a result of the long-lived immunity and adequate anamnestic responses, boosters are not generally required for immunocompetent individuals (10-13). The HBV vaccine has been shown to have a high protective efficacy in population based studies among immunocompetent populations with a protective efficacy of close to 100% in those with HBsAb seroconversion (7;14;15). It is recommended that all patients with HIV be screened for HBV, and if non-immune, receive the recombinant HBsAg vaccine. There are no evidence-based recommendations as to when in the course of their HIV treatment immunization should occur. Among individuals living with HIV, the receipt of the HBV vaccine has not been shown to reduce the risk of subsequent HBV infection (16). The lack of protective efficacy among persons with HIV is due to multiple issues. Seroconversion rates in response to the vaccine are low. In addition, even when seroconversion occurs, there is waning of protective levels of HBsAb and an inadequate anamnestic response (17-22). Prior studies have demonstrated waning of HBsAb levels among persons with HIV after seroconversion, however it is unclear as to the significance of these studies to those living with HIV today, as the majority were done in the era before the introduction of HAART. HAART has had a profound impact on HIV, leading to suppression of HIV viral load, increase in CD4+ cell counts and reduced rates of opportunistic infections. HAART induced suppression of HIV viral replication and resultant immune reconstitution could potentially lead to a longer duration of protection after HBV vaccination (23-27). There is, however, little data in the HAART era to assess the duration of protective immunity. This has led to numerous unanswered questions regarding HBV immunization among persons living with HIV. It is unknown whether HBV vaccination should be deferred until control of HIV replication with HAART or whether HBV vaccination should be repeated once HIV viral suppression has been achieved. The dose of vaccine and whether HBsAb levels should be monitored after seroconversion is uncertain. It is also unknown whether HBV vaccine booster doses are necessary if HBsAb levels fall below the

12 3 threshold of protection, and if provided can boosters induce the development of protective levels of HBsAb (12;21). 2 Study Question The following question will form the basis of the thesis: Among persons living with HIV who received and responded to the HBsAg vaccine with production of protective levels of HBsAb (defined as HBsAb titer of 10mIU/ml) between January 1, 1998 and December 31, 2005, is HIV viral suppression at the time of HBV vaccination associated with persistence of protective levels of HBsAb? The retrospective cohort for the study was constructed and restricted to a time when HAART was readily available and the potential for HIV viral suppression existed. The thesis specifically addresses factors associated with a loss of protective levels of HBsAb and provides valuable information on the anamnestic responses to booster doses of HBV vaccine in those who lose protective levels of HBsAb. This information is integral to the development of recommendations for the effective administration of HBV vaccine to HIV infected individuals.

13 4 Chapter 2 Background The purpose of the chapter is to; 1. Review the epidemiology of HBV among those living with HIV; 2. Evaluate the impact of HBV among those living with HIV; 3. Review the effectiveness of the HBV vaccine among those without HIV; 4. Describe HBV vaccine effects among those living with HIV and the impact HIV viral load suppression has on immune function. 1 Epidemiology of HBV among those living with HIV HBV-HIV co-infection rates vary widely based on risk factors and geography. In the United States and Canada approximately 8-11% of persons with HIV are HBV co-infected (28;29). In Western countries, HBV infection is 10 times more common among HIV infected individuals than those without HIV and is especially common among men who have sex with men (MSMs) and injection drug users. Worldwide, however, the largest population of HBV co-infected individuals are from endemic countries in Africa and the Far East, where co-infection rates are as high as 26% (30;31). With the roll-out of HAART in the developing world, HIV related mortality is expected to decrease and liver-related mortality due to viral hepatitis is expected to increase, mirroring what has happened in the developed world. As a result, effective, evidencebased vaccine strategies for the prevention of HBV will be integral to aborting the additional mortality associated with HBV/HIV co-infection in developing countries. Among immunocompetent adults exposed to HBV, 95% spontaneously recover from acute HBV without the development of chronicity (32). Among HIV infected individuals, however, chronicity develops more frequently, favoring higher HBV disease burden in the HIV community (33;34). Although most HIV negative patients with chronic HBV will not go on to develop hepatic complications during their lifetime, 25-30% will develop serious HBV related disease including end-stage liver disease (ESLD) and hepatocellular carcinoma. Among HIV infected individuals, the natural history of HBV infection is significantly altered. Co-infected 4

14 5 patients have lower rates of HBV inactive states, and more rapid progression to cirrhosis and ESLD (4;5;33-37). 2 Impact of HBV among those living with HIV in the HAART era Since the advent of HAART, there has been a dramatic reduction in the number of deaths related to HIV associated opportunistic infections and malignancy (38). With a longer life span, ESLD related to co-infection with viral hepatitis has emerged as a major cause of mortality among persons with HIV. Thio et al. demonstrated that liver-related mortality among those co-infected with HIV was 14 times higher then in those with HBV alone (4). In a subsequent study by Puoti, the odds of liver related death was nine times higher among those co-infected with HBV (39). These studies and others have clearly established that HBV co-infection is associated with an increased rate of liver-related mortality (40-42). Most patients with HBV co-infection require HBV directed therapy; however, the treatment of chronic HBV in HIV co-infected patients can be difficult compared to the treatment of HBV alone. Interferon therapy in HIV-negative patients is associated with loss of HBV e antigen (HBeAg) in approximately 33% of patients, compared to 10% in HIV infected individuals (43-46). The use of nucleosides (3TC) for the treatment of HBV in those without HIV is associated with HBeAg seroconversion in approximately 30% of patients at one year. however, the durability of the response is limited by relapse in 30 to 50% after cessation of therapy and by the development of drug resistance in two thirds of individuals after 4 years of therapy (47-50). Among co-infected patients, HBeAg seroconversion rates are similar, but resistance occurs more rapidly and more frequently (51-56). After 4 years of 3TC monotherapy, approximately 90% of co-infected patients will develop resistance leading to HBV replication and ongoing liver damage. Newer agents such as tenofovir and entecavir or combination therapy with multiple agents may be associated with lower rates of resistance, yet recent evidence has suggested that HBV viral levels fall more slowly and in some circumstances do not fully suppress with these agents (57-59). In addition, new agents are expensive and are not widely available to the majority of the HIV/HBV co-infected individuals in the developing world where rates of coinfection are highest.

15 6 In HIV negative individuals who develop ESLD due to HBV, orthotopic liver transplantation (OLT) is the treatment of choice. Among persons with HIV, transplantation can be done safely in selected patients and has been adopted in many European and US centres (60-63). In Canada, at the present time, however, OLT is rarely performed and as a result there are few options for those HIV/HBV co-infected patients with ESLD. High liver-related mortality, challenges in the treatment of HBV co-infection and the lack of transplant availability for those with ESLD dictate that prevention rather than treatment is the preferred intervention strategy to address the HBV disease burden among those with HIV. 3 Duration of immunity and the need for boosters among those without HIV The duration of protection conferred after HBsAb seroconversion and the need for booster doses of vaccine are a function of immunologic memory. Among immunocompetent individuals immune memory appears to be long-lived (10;11;13;64-67). A recent study by McMahon et al. demonstrated that among individuals who developed protective levels of HBsAb after vaccination, 78.8% had persistent levels of protective antibodies at 15 years (13). Among the small proportion of immunocompetent individuals who lose protective levels of antibody, anamnestic responses are strong, indicating persistent immunologic memory and protection from HBV infection. (64;68-71). Furthermore, in vitro HBsAg stimulation assays have demonstrated persistent immunologic memory in those with loss of protective levels of HBsAb through the presence of significant B-cell memory (71). Based on these results indicating persistent immunologic memory, the Immunization Practices Advisory Committee (AlCP), The Centre for Disease Control and Prevention (CDC), the National Advisory Committee on Immunization (NACI), as well as the European Consensus Group do not recommend following levels of HBsAb or the use of boosters in hosts with a normal immune system (21;72;73). 4 Protective immunity among individuals living with HIV Traditionally, problems of low seroconversion rates in response to HBV vaccination and waning of immunological memory over time have resulted in concern about the impact of HBV vaccination among those living with HIV. These clinical concerns were confirmed in a recent study demonstrating that the receipt of HBV vaccination among those living with HIV was not

16 7 associated with a reduced risk of HBV (16). Even among those with seroconversion in response to the HBV vaccine there was only a 50% reduction in the risk of developing HBV compared to a vaccine efficacy of close to 100% in immunocompetent individuals (7;16). This lack of protective efficacy of the HBV vaccine, even among those with seroconversion, strongly suggests a problem with immunological memory among persons living with HIV. Specifically, in order to acquire HBV after seroconversion in response to the HBV, vaccine HBsAb levels would need to have fallen below the threshold for protection in the absence of a significant anamnestic response. HBV vaccination in persons with HIV leads to the development of protective levels of HBsAb in approximately 50% of individuals. The response rate, however, is especially poor among those with ongoing HIV viral replication and low CD4+ lymphocyte counts (19;74-92). Due to poor HBV vaccine response rates, several strategies have been developed to improve HBsAb seroconversion. A double dose of HBV vaccine (40mcg instead of the usual 20mcg of Energix B) is recommended by Canadian Guidelines (93). This approach was evaluated in a randomized trial by Fonseca et al. They demonstrated a 25% improvement in seroconversion rates with a double dose of vaccine in those with HIV viral load less than 10,000 copies/ml and CD4+ lymphocyte counts of greater than 350 cells/mm 3 (92). Other consensus guidelines based on expert opinion have suggested delaying immunization until after effective HIV antiviral therapy and immune reconstitution has been achieved in order to increase HBV seroconversion rates (52). Evidence would suggest that this approach would be beneficial, yet response rates even in those with HIV viral suppression and high CD4+ lymphocyte counts are only as high as 60-80% (74;92). This strategy would also place individuals at risk for HBV while waiting for immune reconstitution and subsequent vaccination and would not be applicable for those who do not requiring institution of HAART. Novel vaccination adjuvants such as granulocyte colony stimulating factor (G-CSF) and synthetic oligodeoxynuc1eotides (ODN) containing CpG motifs have shown promise and can lead to improved seroconversion rates and perhaps a longer duration of protective immunity (94;95). Although the use of adjuvants is an exciting development, studies to date have involved only small numbers of subjects and the practicality of these expensive compounds in the developing world, where vaccine demands are high, is questionable.

17 8 Published evidence on the loss of protective immunity after successful vaccination among individuals living with HIV is limited. Available studies from the pre-haart era have demonstrated a loss of protective levels of HBsAb titers in 20-40% of patients at one year. There are, however, several limitations to these data that prevent it from being incorporated into modern day clinical practice for those living with HIV (18;77;85;96-98): 1) all but one study involved hemophiliac populations in an era where contaminated blood products led to frequent HBV exposure and likely immune priming, potentially increasing the observed time to loss of protective levels of HBsAb; 2) immunity was acquired after HBV infection, not induced by HBV vaccination; 3) all studies involved less than 100 HIV positive subjects; 4) plasma derived vaccine was used, likely leading to lower rates of seroconversion compared to the currently used recombinant HBsAg vaccine (99;100); 5) these studies evaluated only the loss of protective levels of HBsAb and did not assess anamnestic responses; and 6) these studies were done in an era without effective HIV therapy involving subjects with active HIV replication and low CD4+ lymphocyte counts. Studies from the HAART era suggest that protective levels of HBsAb are lost in 38.5% to 65% of individuals after one year of follow-up, yet again involved small number of subjects and short duration of follow-up (20;92;101). There is only one study with follow-up data beyond one year. This prospective study demonstrated that 67% of individuals lost protective levels of HBsAb. Post-vaccination titer, older age and lack of response to prior HBsAg vaccination series were predictors of loss of protective levels of HBsAb (102). Studies from the HAART era are limited by small sample size, short duration of follow-up and designed with the intention of evaluating novel vaccination strategies rather than duration of protective immunity. Duration of immune protection after seroconversion in response to the HBV vaccine is a function of HBV specific memory B-cell function, which is significantly altered through active HIV replication (24-26). With the advent of HAART, suppression of HIV plasma viremia is possible, potentially reversing many of the B-cell abnormalities and restoring B-cell responsiveness to immunogens and ex vivo stimuli (23;26). The restoration of immune function with HAART may lead to a significantly prolonged immune protection compared to the results from older studies, with longer time to loss of protective levels of HBsAb and stronger anamnestic responses. Due to the lack of evidence in the HAART era on the duration of protective immunity, the management of the HIV infected patients after vaccine-induced HBsAb seroconversion is

18 9 uncertain. Guidelines and expert reviews suggest measurement of HBsAb at yearly intervals and the provision of boosters to those who lose protective levels (21;52;72;73;93;103). Although this strategy makes intuitive sense, evidence in the HAART era to support this strategy is lacking. This study seeks to clarify these issues in the clinical management of those patients who seroconvert after receiving the HBV vaccination thus leading to a maximization of HBV prevention.

19 10 Chapter 3 Methods The purpose of the chapter is to: 1. Provide an overview of the study design; 2. Describe the study population; 3. Provide definitions of key terms; 4. Define predictor and outcome variables; 5. Describe the statistical approach for analysis. 1 Research Design The study design is an observational, retrospective cohort involving participants selected from one of several HIV clinics in the Greater Toronto Area. The primary cohort is comprised of persons living with HIV who have received the recombinant HBsAg vaccine after their diagnosis of HIV and developed protective levels of HBsAb after vaccination. The primary outcome is loss of protective levels of HBsAb and the main predictor variable is HIV viral load suppression at the time of vaccination. Logistic regression analysis was used to explore independent associations between loss of protective levels of HBsAb and participant characteristics. Participants found to have lost protective levels of HBsAb during follow-up were involved in a prospective component of the study that involved the provision of a booster dose of the recombinant HBsAg vaccine with subsequent evaluation of response. 2 Study Population 2.1 Study Setting Participants meeting eligibility were selected from one of the following HIV clinics in the Greater Toronto Area: 1. Toronto General Hospital (TGH) Immunodeficiency Clinic 10

20 11 2. Toronto Maple Leaf Medical Clinic 3. Sunnybrook Hospital Immunodeficiency Clinic 4. Toronto East General Hospital Immunodeficiency Clinic 5. St. Claire Medical Associates Clinic 6. Medical Clinic at 790 Bay St. The above clinics represent a variety of tertiary and primary care HIV treatment centres that utilize multidisciplinary health teams. These clinics are involved in clinical research and have strong infrastructures that provided resources and support for this project ( ). The primary HIV risk group among the clinics was men who have sex with men (MSMs). Initial centres involved in the study included the Toronto General Hospital Immunodeficiency Clinic and the Toronto Maple Leaf Medical Clinic. The additional centres were added due to smaller than anticipated numbers of enrolled study participants after the first year of the study. 2.2 Study Participants Potential study subjects were identified through review of all medical charts or, if available at the study site, through use of a clinic database. All but one of the study centres had a clinical database. Patients currently being seen in the clinic, those lost to follow-up and those who had died after January 1, 1998 were included in the chart review. The following eligibility criteria were used: Inclusion Criteria HIV positive (HIV-1 viremia or HIV-1/2 antibodies) Documentation of having received at least one dose of recombinant HBsAg vaccine after the time when the potential participant was known to be HIV positive. Last documented dose of HBsAg vaccine between January 1, 1998 to December 31, 2005 (a time period in which HAART was readily available). This did not include booster doses of HBsAg vaccine. A booster was defined as the provision of a single dose of HBsAg vaccine after HBsAb titers have fallen to <10mIU/ml after previous seroconversion to a preceding HBsAg vaccination series.

21 12 HBsAb seroconversion (defined as a HBsAb titer of 10mIU/ml on a serum sample taken after the last dose of recombinant HBsAg vaccine in an individual with pre-vaccine HBsAb titer of < 10mIU/ml) Exclusion Criteria Chronic HBV infection (HBsAg positive or HBV DNA detectable) Receipt of the recombinant HBsAg vaccine prior to HIV diagnosis All potential subjects, other than those patients who had died, were approached for consent (see Appendix A). Patients currently being seen in the clinic were consented in person at their next clinic appointment by a research assistant not directly involved in the patient s care. Patients who were lost to follow-up (not seen in clinic in the preceding 12 month period) were contacted by mail to their last known address and were asked to contact the research coordinator by phone/mail for consent (see Appendix B). Those patients who contacted the coordinator were consented in person. If patients lost to follow-up did not contact the research coordinator in a three month period after the original letter was mailed requesting their participation, they were enrolled in the study. Those patients who had died were screened and enrolled if they met eligibility criteria. The above outlined process for enrolment was approved by the REBs at all participating centres. Enrolment was halted when the estimated sample size was reached. The decision to enroll participants who were lost to follow-up or who had died without formal consent was initiated in order to ensure we did not introduce selection bias to our study. We assumed those lost to follow-up or who died would be different than the population of patients still actively being seen in the clinic, particularly with respect to important predictor variables such as viral load suppression. Due to the potential risk selection bias would have introduced to the study s validity and the low risk nature of the study we felt this was ethically justifiable. Seroconversion status was not available for all participants through review of existing clinical records. Among these potentially eligible participants, seroconversion status was assessed through retrospective evaluation of stored serum samples from prior HIV viral load testing. From 1997 to present, serum samples from HIV viral load testing were frozen and saved as part of the viral load testing program in Ontario if the HIV viral load was greater than or equal to 50 c/ml. The study investigators have used saved serum samples from HIV viral load testing in a similar fashion in prior studies with success (105; ). Participants were excluded if seroconversion

22 13 could not be demonstrated through either chart review or testing of saved HIV viral load specimens. All HBsAb testing was done by the Toronto Medical Laboratories or at the Toronto Public Health Laboratory under the supervision of one of the study investigators (Dr. Tony Mazzulli) with the use of the automated Axsym (Abbot Systems, Canada) system. The Axsym system uses an enzyme-linked immunoreacting antigen absorbed onto microparticles to detect the primary interaction of antibody. Titers were expressed as miu/ml. Loss of protective levels of HBsAb were determined through testing of an individual's most recent stored frozen serum sample from HIV viral load testing or testing of new clinical samples. Individuals with loss of protective levels of HBsAb were asked to return to clinic to receive a booster dose of the recombinant HBsAg vaccine. The booster consisted of a 40mcg IM dose of Engerix. This is considered to be a double dose of the vaccine as it is twice the usual dose provided to immunocompetent individuals. After booster doses of vaccine were administered individuals were asked to return to clinic after at least one month and up to one year for determination of HBsAb status. Information on variables of interest was abstracted from patients charts. Missing variables were addressed by utilizing alternative data sources (i.e. the patient s family physician s chart). Patients were assigned a study number after enrolment, which was used to identify them through the duration of the study. Only study investigators had access to the master list referencing patients' chart medical record number to study number. The master list was kept in a secure, locked location in the Toronto General Hospital Immunodeficiency Clinic and subsequently destroyed at study completion. Data forms (see Appendix C) were checked for completeness and logical errors before being double-entered into a password protected Microsoft Access database. Data forms will be destroyed 5 years after study publication. Data was stored confidentially (password protected computer; back-up hard copies kept in a locked filing cabinet in the Toronto General Hospital Immunodeficiency Clinic). Study data was accessible only to study staff. The study was approved by the Research Ethics Board of participating centres as well as the University of Toronto.

23 14 3 Definitions Important study variables were defined as follows: 3.1 Protective levels of HBsAb A protective level of HBsAb was defined as an HBsAb level of greater than or equal to 10mIU/mL. This cut-off value for protection was determined from prevention studies of passive immunization and the plasma derived vaccine. These studies correlated HBsAb values of 10mIU/mL with protection from clinically significant HBV infection after exposure (6-8). Loss of protective levels of HBsAb The decrease in HBsAb levels from greater than or equal to 10mIU/ml to a value less than 10mIU/ml. 3.2 Anamnestic Response An increase in HBsAb levels to greater than or equal to 10mIU/ml after receiving a booster dose of the recombinant HBsAg vaccine from levels of HBsAb of less than 10mIU/ml prior to receipt of the booster. The post-booster HBsAb levels were determined at least one month, yet no greater than 12 months post-booster dose of HBV vaccine. 3.3 Loss of protective immunity Loss of protective immunity was defined as a composite outcome including loss of protective level of HBsAb and absence of an anamnestic response in participants who have received a booster dose of the HbsAg vaccine. 3.4 HIV viral load suppression HIV viral suppression was defined as having an HIV viral load of 50 copies/ml on PCR testing (Chiron 2.0 Assay)

24 HIV viral load blip An increase in a participant s HIV viral load, from previously suppressed values (less than 50 copies/ml), to a value of 50 copies/ml to 500 copies/ml on a single measurement with subsequent return to suppressed values on the next measured HIV viral load. 3.6 Post-vaccination titer Level of HBsAb taken from 1 to 6 months after the last dose of a HBV vaccine series was administered. 4 Key Predictor and Outcome variables 4.1 Outcome: Loss of protective levels of HBsAb during followup The outcome of interest in this study was loss of protective levels of HBsAb during follow-up. This was determined by measuring the HBsAb at the last clinical evaluation or by testing the HBsAb through utilization of saved HIV viral load specimens. Loss of protective levels of HBsAb is an important clinical outcome as it may be associated with risk of chronic HBV acquisition with subsequent HBV exposure among immunocompromised populations. 4.2 Primary predictor variable of interest The primary predictor variable of interest for this study was HIV viral load suppression. HIV viral suppression was evaluated in two ways: 1) whether HIV viral load was suppressed at the time of the last dose of vaccine and 2) proportion of time spent with HIV viral load suppression from time of last dose of vaccine to outcome determination. Participants with HIV viral load blips were still considered to have HIV viral load suppression. HIV viremia is associated with a large number of immune deficiencies, one of which includes poor CD4+ cell response to antigen challenge. HIV viral load suppression with antiretroviral medications leads to the reversal of many of the immune defects associated with HIV(23-27).

25 16 HIV viral load suppression was evaluated in the above two ways in an attempt to differentiate whether HIV viral load suppression at time of vaccination was more important than HIV viral load suppression after vaccination. This has direct relevance to vaccination strategies when clinically evaluating a person living with HIV. Specifically, should HBV vaccination be delayed until HIV viral load is suppressed or can vaccination be provided immediately with subsequent HIV viral load suppression thereafter? 4.3 Other factors potentially associated with loss of protective levels of HBsAb 1) Receiving at least 3 doses of the recombinant HBsAg vaccine. The recombinant HBsAg vaccine is suggested to be given at 0, 1 and 6 months, however, many patients will receive less than the recommended three doses. Patients who receive at least 3 doses of the recombinant HBsAg vaccine were compared to those who received one or two doses. The number of HBsAg vaccine doses was determined through chart review and in situations where the number of doses was unclear, additional data sources were utilized to determine the number of HBsAg vaccine doses received. 2) CD4+ cell count. Lower CD4+ cell counts are associated with poor humoral responses to antigen challenge (24). HBV vaccine studies in HIV positive individuals have demonstrated low CD4+ cell counts to be associated with low seroconversion rates(19;74-90;92;115). The association between low CD4+ cell counts and loss of protective immunity to HBV has not been examined. The CD4+ cell count will be evaluated in several ways: 1) CD4+ cell count at time of receipt of the last dose of recombinant HBsAg vaccine; 2) CD4+ cell count greater than 350 cells/mm 3 at time of receipt of the last dose of recombinant HBsAg vaccine; 3) CD4+ cell count greater than 200 cells/mm 3 at time of receipt of the last dose of recombinant HBsAg vaccine; 4) time spent with CD4+ cell count above 350 cells/mm 3 from time of last dose of HBV vaccine to outcome determination; and 5) time spent with CD4+ cell count above 200 cells/mm 3 from time of last dose of HBV vaccine to outcome determination. The CD4+ cell count was evaluated in different ways to evaluate potential associations between CD4+ cell count and loss of protective levels of HBsAb at different CD4+ thresholds at both the time of vaccination and during follow-up.

26 17 The CD4+ cell count cut-offs were chosen based on the current treatment guidelines suggesting initiation of HAART at a CD4+ cell count of 350 cells/mm 3 and the Centre for Disease Control (CDC) definition of AIDS at a CD4+ cell count of 200 cells/mm 3 (116) 3) Immunosuppressive conditions. Renal failure (creatinine > 2 x upper limit of normal or renal replacement therapy), malignancy requiring systemic therapy, alcohol abuse and diabetes mellitus requiring treatment have been linked to poor HBV vaccine response likely related to their impact on T-cell help and B-cell function ( ). The presence of any of these conditions, at any time between first dose of vaccine and outcome determination, was considered to be an immunosuppressive condition. 4) Hepatitis C virus (HCV). Those with HIV and HCV may be more susceptible to HBV infection even after receiving the vaccine (121). Due to the chronic nature of HCV infection among persons with HIV, any HCV-antibody positive test prior to outcome determination was considered to indicate the presence of HCV. 5) Time elapsed from date of vaccination to outcome determination. Prior studies from the pre-haart era demonstrated that HBsAb levels decline over time. 6) Age at time of vaccination and Gender 5 Statistical Analysis 5.1 Primary Analysis Due to the low probability of HBsAb loss during the first year of follow-up, only participants with duration of follow-up greater than 12 months were included in the analysis. The association between HIV viral load, as well as other potential factors with loss of protective levels of HBsAb was first compared between individuals with and without loss of protective immunity using the Chi Square test or Fisher s Exact test for categorical variables and the two sample t-test or Mann Whitney Test for continuous variables, according to whether or not the distribution was normally distributed. The specific CD4+ cell variable was selected for the multivariable logistic regression model based on which variable was best associated with HBsAb loss in univariate analysis.

27 18 Potential correlation and interactions between predictor variables and the loss of protective levels of HBsAb were assessed graphically. Thereafter, due to the multiplicity of the factors potentially associated with the loss of protective levels of HBsAb, multiple logistic regression models were used to estimate the effect of virologic suppression on the probability of persistence of protective levels of HBsAb after controlling for other covariates. Two multivariable models were constructed. One utilizing the proportion of time spent with HIV viral load suppression from time of last dose of vaccine to outcome determination and the other using viral load suppression at time of last dose of vaccine. The HIV viral load variables were run in two separate models due to the high degree of correlation between them. Additional factors included in the analysis were age, gender, CD4+ count, HCV infection, time from vaccination to outcome determination, and the presence/absence of an immunosuppressive condition. Correlation among variables was assessed to ensure variables could be evaluated independently. Due to low numbers of participants with HCV antibody positivity and immunosuppressive conditions, these variables were removed from the final model. The final multivariable model included one of the two HIV viral load suppression variables, CD4+ cell count greater than 350 cells/mm 3 at time of vaccination, age, gender, duration of follow-up and receipt of at least 3 doses of the vaccine. 5.2 Secondary Analyses A secondary analysis was planned to evaluate factors associated with loss of protective immunity (see definitions above). As all of the participants who lost protective levels of HBsAb and received a booster dose of vaccine developed protective immunity, this analysis could not be performed as there was no comparator group. The response to the booster dose of vaccine among those with loss of protective levels of HBsAb was described as a percentage of those individuals with the development of protective levels of HBsAb after receiving the booster.

28 Sample size and power Initial sample size calculations indicated that if 50% of the study population had HIV viral load suppression, with 80% power and a significance level of 0.05, the following table provides the required sample sizes for varying effect sizes. We assumed that 60% of those participants with HIV viral load suppression lost protective levels of HBsAb compared to 80% without HIV viral load suppression. A total sample size of 162 (81 per group) was required. Table 1: Sample Size per group with varying assumptions for loss of protective levels of HBsAB. Effect Size of Interest (p 2 -p 1 ) P 1 15% 20% 25% * * Assumes p 2 =0.99 We used the control arm of a recent study by Cooper et al. to justify our sample size. In this study, all individuals had suppression of HIV viral load and approximately 20-30% lost protective levels of HBsAb at one year (94). We assumed an ongoing linear decline in HBsAb titers arriving at our estimate of 60% of individuals with HIV viral load suppression losing protective levels of HBsAb.

29 20 Chapter 4 Results The objectives of this chapter are to: 1. Describe the cohort; 2. Compare baseline characteristics of those with and without viral load suppression at time of vaccination; 3. Determine the impact of HIV viral load suppression on persistence of protective levels of HBsAb; 4. Describe response to booster doses of vaccine in those with loss of protective levels of HBsAb; 5. Describe relationship between post-vaccination HBsAb titers and loss of protective levels of HBsAb. 1 Description of the cohort A total of 298 potential participants were approached for consent at all six centres participating in the trial. The majority of the participants were enrolled at the University Health Network Immunodeficiency Clinic and the Maple Leaf Medical Clinic and were actively being followed in clinic. Of the 298 participants approached, 250 provided informed consent. Of the 250 consented participants, 164 met eligibility criteria after confirmation of seroconversion status. The majority of patients were excluded due to either lack of seroconversion or inability to confirm seroconversion with testing of saved HIV viral load specimens. Of the 164 study participants, 152 had greater than 12 months of follow-up and were included in subsequent analysis. The baseline characteristics of the 152 study participants with greater than 12 months of followup are presented in Table 2. The average study participant was a 46 years old, MSM without advanced HIV (mean CD4+ cell count of 535 with 52% having HIV viral load suppression at the time of vaccination). The median duration of follow-up was 43 months with an interquartile range of months. 20

30 21 Table 2: Patient demographic and clinical characteristics at time of vaccination Variable Age (years) Mean 41.8 (8.1) Gender Male 131 (86.2%) Clinical Status Active 141 (92.7%) Lost to follow-up 7 (4.6%) Deceased 4 (2.6%) Self-Reported Risk Group Men who have sex with men (MSM) Heterosexual Vertical Blood transfusion/nosocomial IVDU Study Centre TGH Maple Leaf TEGH Sunnybrook St. Claire 790 Bay 113 (74.3%) 37 (24.3%) 0 3 (2.0%) 3 (2.0%) 46 (30.3%) 50 (32.9%) 9 (5.9%) 18 (11.8%) 23 (15.1%) 6 (3.9%) 0.08, , 0.35 HCV Ab positive (n=150) 3 (2.0%) Immunosuppressive condition 6 (4.0%) Duration follow-up in months 43, Received at least 3 doses of vaccine 84 (55.3%) HIV VL < 50c/ml at time of vaccination 79 (52.0%) Proportion of time of follow-up spent with HIV VL <50c/ml 0.63, 0.42 CD4+ cell count at time of vaccination 535, 286 CD4+ cell count > 200 cells/mm 3 at time of vaccination 139 (91.4%) Proportion of time of follow-up spent with CD4+ cell count < 200 cells/mm 3 CD4+ cell count > 350 cells/mm 3 at time of vaccination 112 (73.7%) Proportion of time of follow-up spent with CD4+ cell count < 350 cells/mm 3 Mean, Standard deviation Median, Interquartile range Abbreviations: IVDU=Intravenous Drug User, TGH = Toronto General Hospital Immunodeficiency Clinic, Maple Leaf= Toronto Maple Leaf Medical Clinic, TEGH = Toronto East General Hospital Immunodeficiency Clinic, Sunnybrook = Sunnybrook Hospital Immunodeficiency Clinic, St. Claire = St. Claire Medical Associates Clinic, 790 Bay = Medical Clinic at 790 Bay Street, HCV Ab= Hepatitis C Virus antibody, VL= viral load.

31 22 2 Baseline characteristics according to HIV viral load suppression at time of vaccination Those with HIV viral load suppression at the time of vaccination differed from those without HIV viral load suppression in several aspects (Table 3). Looking at baseline demographics, those with HIV viral load suppression were on average 5 years older at time of vaccination and had 11 months shorter duration of follow-up than those without HIV viral load suppression. Chronic HCV was more common among those without viral suppression while immunosuppressive conditions other than HIV were more common among those with HIV viral load suppression. This was based, however, on the low numbers of individuals in the cohort with both of these conditions. Not surprisingly, there was a non-significant trend towards higher CD4+ve cell counts among those with viral load suppression.

32 23 Table 3: Characteristics among those with and without HIV viral load suppression at time of receipt of last dose of recombinant HBsAg vaccine (n = 152). Variable HIV VL suppression (n=79) HIV VL not suppressed (n =73) p value Age (years) 44.0 (7.4) 39.5 (8.1) <0.001 Male Gender 66 (83.5%) 65 (89.0%) 0.33 * Study Centre 0.64 UHN Maple Leaf TEGH Sunnybrook St. Claire 790 Bay 22 (27.8%) 26 (32.9%) 6 (7.8%) 9 (11.4%) 11 (13.4%) 5 (6.3%) 24 (32.9%) 24 (32.9%) 3 (4.1%) 9 (12.3%) 12 (16.4%) 1 (1.4%) HCV Ab positivity (n=150) 0 3 (4.1%) 0.11 Immunosuppressive condition 6 (7.6%) Duration follow-up φ 38 (28-58) 49 (34-67) 0.03 # Received at least 3 doses of 44 (55.7%) 40 (54.8%) 0.91 * vaccine CD4+ cell count at time of 572 (337) 494 (213) 0.09 vaccination (cells/mm 3 ) CD4+ cell count > 200 cells/mm 3 at time of vaccination Proportion of time of followup spent with CD4+ cell count < 200 cells/mm 3 ) CD4+ cell count > 350 cells/mm 3 at time of vaccination Proportion of time of followup spent with CD4+ cell count < 350 cells/mm 3 ) 72 (91.1%) 67 (91.8%) 0.89 * 0.07 (0.20) 0.08 (0.20) (73.4%) 54 (74.0%) 0.94 * 0.23 (0.37) 0.26 (0.34) 0.61 Student s t test * Chi-Square Fisher s Exact Test # Wilcoxon Rank Sum Test Mean, Standard deviation φ Median, Interquartile range Abbreviations: IVDU=Intravenous Drug User, TGH = Toronto General Hospital Immunodeficiency Clinic, Maple Leaf= Toronto Maple Leaf Medical Clinic, TEGH = Toronto East General Hospital Immunodeficiency Clinic, Sunnybrook = Sunnybrook Hospital Immunodeficiency Clinic, St. Claire = St. Claire Medical Associates Clinic, 790 Bay = Medical Clinic at 790 Bay Street, HCV Ab= Hepatitis C Virus antibody.

33 24 3 HIV viral load suppression at time of vaccination and loss of protective HBsAb titers 3.1 Univariate analysis of factors associated with loss of protective levels of HBsAb The only factor significantly associated with loss of protective levels of HBsAb in univariate analysis was HIV viral load suppression at time of last dose of vaccine (Table 4). Individuals with viral load suppression at time of vaccination lost protective levels of HBsAb at a frequency of 16.5% compared to 38.4% among those without HIV viral load suppression at the time of their last dose of vaccine. The proportion of time spent during follow-up with a suppressed HIV viral load was of borderline significance, with a trend toward a higher proportion of time during follow-up spent with HIV viral suppression among those without loss of HBsAb (p = 0.09).