Alternative Pneumococcal Vaccination Schedules for Infants in Fiji and Pneumococcal Epidemiology

Transcription

1 Alternative Pneumococcal Vaccination Schedules for Infants in Fiji and Pneumococcal Epidemiology Dr Fiona Mary Russell Submitted in total fulfillment of the requirements of the degree of Doctor of Philosophy 11 th October, 2010 Department of Paediatrics Faculty of Medicine, Dentistry, and Health Sciences The University of Melbourne

2 i ABSTRACT This thesis documents the pneumococcal disease burden and the results of a Phase II pneumococcal vaccine trial in the low middle income country, Fiji. The overall objective was to gather sufficient evidence for the Fiji Ministry of Health to decide whether to introduce the pneumococcal vaccination into its national schedule and define an appropriate and affordable vaccination strategy. The nasopharynx is the main reservoir for pneumococci and plays an important role in the spread of the organism. Studies of nasopharyngeal carriage offer insights into the pneumococcal disease burden in a community, particularly for potential serotypes which may cause pneumonia, and are a convenient way of determining the level of antibiotic resistance among pneumococcal isolates circulating in a population. The first study, a crosssectional pneumococcal nasopharyngeal carriage survey of healthy children aged 3 13 months, was undertaken to document the prevalence of pneumococcal nasopharyngeal carriage, risk factors for carriage, serotypes and antimicrobial susceptibility patterns of carried pneumococci in healthy young children in Fiji (Chapter 3). Pneumococcal nasopharyngeal carriage was common in Fijian children. Penicillin resistance was documented for the first time, and, as a result, first-line treatment for meningitis was altered. A low proportion of carriage serotypes were included in the 7-valent pneumococcal conjugate vaccine. Invasive pneumococcal disease is an important cause of morbidity and mortality, particularly in the very young and the elderly. The introduction of the 7-valent pneumococcal conjugate vaccine in the national immunisation schedule in the USA has resulted in an impressive reduction in infant invasive pneumococcal disease. In addition, the vaccine has had a more than expected herd immunity effect on invasive pneumococcal disease in the elderly and other age groups. Chapter 4 reports on a study that aimed to document age-specific burden of invasive pneumococcal disease including clinical syndromes, underlying conditions, serotype distribution, and the potential protection against invasive pneumococcal disease and chest X-ray confirmed pneumonia by 7-valent pneumococcal conjugate vaccine in Fiji. The annual invasive pneumococcal disease incidence was comparable to countries of similar socioeconomic status. Being indigenous Fijian was an independent risk factor for disease. Underlying conditions were common and the case fatality rate was high particularly in the elderly population. For every 1,930 and 128 infants vaccinated, one death and one case

3 ii respectively, would be prevented in those <5 years, by introduction of universal immunisation with the 7-valent conjugate vaccine. A Phase II vaccine trial was undertaken to document the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination regimens combining one, 2, or 3 doses of 7-valent pneumococcal conjugate vaccine in infancy (Chapters 5 to 10). In order to broaden the serotype coverage, the additional benefit of a booster of 23-valent pneumococcal polysaccharide vaccine at 12 months of age was also assessed. To address the theoretical concerns of hyporesponsiveness to 23-valent pneumococcal polysaccharide vaccine following re-challenge, the immunological responses at 17 months of age to a small challenge dose of 20% of 23-valent pneumococcal polysaccharide vaccine (mpps) in children who had or had not received the 23-valent pneumococcal polysaccharide vaccine at 12 months of age was undertaken. The immunogenicity following a 2 or 3 dose 7-valent pneumococcal conjugate vaccine primary series was similar for many serotypes. A single 7-valent pneumococcal conjugate vaccine dose would offer protection in the first 12 months of life for many serotypes. The one or 2 dose 7-valent pneumococcal conjugate vaccine schedules induced immunologic memory, with memory responses following 23-valent pneumococcal polysaccharide vaccine being most profound for children who had received only a single dose of 7-valent pneumococcal conjugate vaccine previously, compared with the 2 or 3 dose groups. Following the 23-valent pneumococcal polysaccharide vaccine booster, there were significant responses for all 23 serotypes which persisted for at least 5 months following vaccination. However despite higher antibody concentrations at 17 months in children who had received 23-valent pneumococcal polysaccharide vaccine at 12 months, the response to a re-challenge was poor to all 23 serotypes compared to children who did not receive the 12 month 23-valent pneumococcal polysaccharide vaccine. This indicates immunological hyporesponsiveness or non-responsiveness. This effect occurred regardless of pre-mpps antibody levels and prior 7-valent pneumococcal conjugate vaccine exposure. The addition of 23-valent pneumococcal polysaccharide vaccine at 12 months had no impact on carriage, despite the substantial boosts in antibody levels observed and despite significantly higher opsonophagocytic activity and antibody avidity comparing pre- and post-levels. In summary, a substantial burden of pneumococcal disease in Fiji was found. The 7-valent pneumococcal conjugate vaccine would provide limited coverage of invasive disease compared to its use in affluent countries. Two doses of 7-valent pneumococcal conjugate

4 iii vaccine have similar immunogenicity as 3 doses although a single dose still provides some protection. The 23-valent polysaccharide vaccine booster was found to be immunogenic but re-challenge resulted in hyporesponsiveness. Further research evaluating the potential of reduced dose schedules using the newer conjugate vaccines with an early conjugate booster would be recommended.

5 iv DECLARATION This is to certify that 1. the thesis comprises only my original work towards the PhD except where indicated in the Preface, 2. due acknowledgement has been made in the text to all other materials used, 3. the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies, and appendices. Name: Fiona Russell Signature: Date: 11 th October, 2010

6 v PREFACE Abstract: This chapter is entirely my work under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. Chapter 1: Literature Review This chapter is entirely my work under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. Chapter 2: Extended Materials and Methods section I was responsible for writing the bulk of this chapter under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. Prof Kim Mulholland outlined the initial concept of the design of the study. Expert advice was sought from Porter Anderson, Brian Greenwood, George Siber, and DMID NIH personnel for various aspects of the study. The fieldwork SOPs were written by Sam Colquhoun, Jane Nelson, and Vanessa Johnson, under my direction and supervision. The laboratory ELISA and avidity SOPs and methods were written by Anne Balloch and Paul Licciardi. The OPA methods were written by Rob Burton and Moon Nahm, University of Alabama at Birmingham, Birmingham, Alabama, USA. The microbiology laboratory SOPs were written by Chris Pearce (formerly from the Royal Children s Hospital, Melbourne) and Shirley Warren (Westmead Hospital). The statistics section, including sample size calculations were written by Graham Byrnes. Chapter 3: Pneumococcal nasopharyngeal carriage and patterns of penicillin resistance in young children in Fiji I was responsible for the design of the study under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the protocol, submitted the study to the ethics committee for approval, and supervised the fieldwork. Senibua Ketawai, the laboratory technician, processed the specimens. Dr Viema Kunabuli and Mabel Taoi collected the specimens. Sharon Biribo and Anna Seduadua performed the serotyping. I analysed the data, wrote the first draft of the manuscript with input from the co-authors on the published paper: JR Carapetis, S Ketawai, V Kunabuli, M Taoi, S Biribo, A Seduadua, EK Mulholland.

7 vi Chapter 4: Epidemiology of Invasive Pneumococcal Disease in Fiji: the potential impact of pneumococcal conjugate vaccine I was responsible for the design of the study under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the protocol, submitted the study to the ethics committee for approval, and reviewed all the medical records. Anna Seduadua and Reginald Chand processed and stored the isolates. Catherine Satzke processed the isolates for shipment for serotyping. Shahin Oftadeh and Prof Lyn Gilbert were responsible for serotyping the isolates. I analysed the data, wrote the first draft of the manuscript with input from the co-authors on the published paper: JR Carapetis, L Tikoduadua, R Chandra, A Seduadua, C Satzke, J Pryor, E Buadromo, L Waqatakirewa, EK Mulholland. Chapter 5: Immunogenicity Following One, Two, or Three Doses of the 7-valent Pneumococcal Conjugate Vaccine Chapter 6: Safety and Immunogenicity of the 23-Valent Pneumococcal Polysaccharide Vaccine at 12 months of age, following One, Two, or Threes Doses of the 7-valent Pneumococcal Conjugate Vaccine in Infancy Chapter 7: Hyporesponsiveness to Re-challenge Dose Following Pneumococcal Polysaccharide Vaccine at 12 Months of Age, a Randomized Controlled Trial Chapter 8: Serotype-specific avidity is achieved following a single dose of the 7-valent pneumococcal conjugate vaccine, and is enhanced by 23-valent pneumococcal polysaccharide booster at 12 months For Chapters 5-8 I was responsible for writing the original protocol under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the ethics submission, was responsible for the annual ethics report submissions, and I supervised the fieldwork. The assays were performed by Anne Balloch, Paul Licciardi, and Mimi Tang. I was responsible for cleaning and analysing the data under the supervision of Yin Bun Cheung and Prof Kim Mulholland. I wrote the first draft of the manuscripts with input from the co-authors: PV Licciardi PV, A Balloch A, V Biaukula V, L Tikoduadua L, JR Carapetis JR, J Nelson, AWJ Jenney, L Waqatakirewa, S Colquhoun, YB Cheung, MLK Tang, EK Mulholland. Funding was provided by the U.S. NIAID (grant number R01 AI 52337) and the Australian National Health and Medical Research Council. Pneumovax TM was kindly donated by CSL Biotherapies, Australia. The co-administered Tritanrix TM -HepB TM and Hiberix TM vaccines were kindly donated by GlaxoSmithKline. Clinicaltrials.gov number NCT

8 vii Chapter 9: Opsonophagocytic Activity Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Infant Primary Series and 23-valent Pneumococcal Polysaccharide Vaccine at 12 Months of Age I was responsible for writing the original protocol under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the ethics submission, was responsible for the annual ethics report submissions, and I supervised the fieldwork. The assays were performed by Rob Burton and Moon Nahm. I was responsible for cleaning and analysing the data under the supervision of Yin Bun Cheung and Prof Kim Mulholland. I wrote the first draft of the manuscripts with input from the co-authors: JR Carapetis, RL Burton, J Lin J, PV Licciardi, A Balloch, L Tikoduadua, L Waqatakirewa, YB Cheung, MLK Tang, MH Nahm, EK Mulholland. In addition to the funding stated above, funding was also provided by U.S. NIAID grant number N01 AI Chapter 10: Pneumococcal Nasopharyngeal Carriage Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Schedule in Infancy and a 23-valent Pneumococcal Polysaccharide Vaccine Booster, a Randomised Controlled Trial I was responsible for writing the original protocol under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the ethics submission, was responsible for the annual ethics report submissions, and I supervised the fieldwork. The assays were performed by Anna Seduadua, Reginald Chandra, Catherine Satzke and the laboratory staff at the Centre for International Child Health (Murdoch Children s Research Institute, Royal Children s Hospital and Department of Paediatrics, University of Melbourne), Shahin Oftadeh, and Prof Lyn Gilbert. I was responsible for cleaning and analysing the data under the supervision of Yin Bun Cheung and Prof Kim Mulholland. I wrote the first draft of the manuscripts with input from the co-authors: JR Carapetis, C Satzke, L Tikoduadua, L Waqatakirewa, R Chandra R, A Seduadua, S Oftadeh, YB Cheung, GL Gilbert, EK Mulholland. Chapter 11: Conclusion This chapter is entirely my work under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis.

9 viii ACKNOWLEDGEMENTS Study Participants I am indebted to all the parents in the Nausori, Valelevu, Makoi, and CWMH catchment who consented for their babies to participate in the studies and came to the clinics so many times for visits and procedures for 2 years. Without these willing parents these studies would not have been possible and for which I am eternally grateful. The FiPP Team I wish to thank all the dedicated FiPP team who tirelessly worked for up to 6 years, enthusiastically undertook what was requested of them, worked so well as a team, and were keen to learn as much as they could whilst on the project. The many FiPP study nurses who enthusiastically dedicated themselves to the study and cared for the parents and children so well: Tania Ah Kee, Felisita Tupou Ratu, Elina, Priya Frances, Liti, Agnes Rounds, Amelia Wara, and Mabel Taoi. It had been a pleasure to work closely with Dr Viema Biuakula who worked so hard and graciously, completed her own study, a Masters in Public Health, married and started her own family, and launched her own career in public health surveillance in the Ministry of Health. For Mere Vakacegu who was our first employee, starting as the cleaner and within a short time became an expert in databases and database design. For Robert Cabemaiwasa, who for a long time tolerated being our only male employee and excelled in transporting specimens, entered countless CRFs year after year without a complaint, and occasionally indulged us with his magnificent muffins. For Simi Sokiqele and his dedication to detail, precision, his delightful letters, and interesting insights into Fiji life and culture. For Senibua Kataiwai, Anna Seduadua and Reginald Chandra who worked ad naseum, tucked away in the lab processing and storing the many thousands of specimens. For Taraifina Saro and Sharonika Chand who smoothed the way through immigration, managed the office, and made sure we never ran out of pens and that the bills were paid. I have been extremely fortunate to have 3 study co-ordinators who I am very grateful for their hard work, expertise and tireless work over 6 years. I thank Sam Colquhoun whose organisation, hard work, expertise in GCP and setting up clinical trials provided the foundation for a successful study. Jane Nelson whose uncompromising attention to detail, organisation, seemingly non-stop ability to take on more work, ability and willingness to sort out field work issues, and ongoing supervision provided such an excellent basis for all the

10 ix quality research undertaken. For Kathryn Bright who quickly stepped into such big shoes and co-ordinated the fieldwork to such high quality and saw the successful completion for the study with cheer and diplomacy. I thank her for her friendship. For Beth Temple who provided expert advice in database management and introduced me to the efficient magical stata loops for analysing the voluminous amount of data. Fiji Ministry of Health We have had a very close relationship with the Ministry of Health and wish to thank them enormously for collaborating and hosting our project, providing valuable space for us in the Ministry of Health clinics, providing advice on logistical issues, smoothing the way for administrative issues, and for the acceptance we have had for undertaking the first clinical trial in Fiji. Dr Lepani Waqatakirewa, the former Permanent Secretary the Fiji Ministry of Health, was an investigator on the project, was involved from the project s inception, and I have appreciated his support and wise counsel. I had the pleasure of working closely with Dr Lisi Tikoduadua, former Head of Paediatrics at the Colonial War Memorial Hospital, a tremendous advocate for child health for Fiji and the region. Lisi was involved in our project from its very inception and has always offered support for me personally and for our project. I have enjoyed her humour and philosophical way of dealing with delays and obstacles. She embraced research, made us welcome, and generously made office space available for us within her department. To Dr Joe Kado, the current Head of Paediatrics, who also provided support, showed much interest, and valued our work. To Dr Sala Saketa, the current Permanent Secretary for Health and Dr Josaia Samuela, Family and Reproductive Health at the Fiji Ministry of Health have been very supportive and interested the project. Dr Eka Buadromo, Head of the Department of Pathology at Colonial War Memorial Hospital, generously provided space within the microbiology laboratory and was always supportive and involved in our work. I wish to thank all of the staff members within the microbiology laboratory particularly Shalini Singh and Parmod Kumar. I wish to thank Senibua Ketawai for establishing the lab work and joining the quest in search of a reliable source of animal blood. I wish to thank the serology laboratory staff for being so accommodating. I would like to thank the clinical staff on the paediatric wards of the hospital. In particular I would like to thank Matron Balawa who helped point me in the right direction when all we started with were 2 empty offices.

11 x I would also like to thank the staff in the Medical Records department, particularly Niumia Hicks, the General Manager of Community Health within the CentEast Health Division, Dr Solomone Qaranivalu, and Idrish Khan, Chief Finance Officer at the Ministry of Health, who was always so accommodating regarding the financial arrangements of the project. Fiji School of Medicine I wish to thank Dr Jan Pryor for ongoing support and being a tremendous advocate for the value of our research. I wish to thank Prof Rob Moulds for making himself available and being interested in our work. I wish to thank Sharon Biribo for explaining the intricacies of microbiology to a novice, joining the quest to find a reliable source of animal blood, assisting the establishment of our pneumococcal lab work, and undertaking some of the serotyping. I wish to thank Vicki Chand whose energy, enthusiasm, and positive attitude rubbed off on those around her and for establishing our databases, and Dr Rosa Sa aga who was involved in our project from its inception and was always very supportive. Koronivia Veterinary Research Station In search of a reliable source of animal blood to make blood agar I am indebted to the kind assistance of Fiji s former chief veterinarian (Fiji s own James Herriot) and the staff of Koronivia Veterinary Research Station who took me on a tour of the farms in Fiji, bled horses and sheep for us, allowed us to experiment with different blood collection techniques before arranging for the purchase of our own FiPP sheep, which they housed, took care, and bled as required. Pneumococcal Laboratory, Murdoch Childrens Research Institute, Royal Children s Hospital, Melbourne I am grateful to Anne Balloch for establishing a first class Pneumococcal laboratory and Dr Paul Licciardi for completing the extraordinary job of churning through tens of thousands of assays over the many years of the project with such good humour, willingness, and efficiency. I am also grateful for their technical immunological insights with the write up. I wish to thank Prof Mimi Tang for her technical oversight and helpful comments, and the laboratory support from Amy Bin Chen and Timothy Gemetzis.

12 xi Pneumococcal Reference Laboratory, Centre for Infectious Diseases & Microbiology, ICPMR, Westmead, NSW I am grateful to Prof Lyn Gilbert and Shahin Oftadeh for undertaking the task of serotyping all our isolates and solving the technical issues surrounding the serotyping. Bacterial Respiratory Pathogen Reference Laboratory, University of Alabama at Birmingham, USA I wish to thank Prof Moon Nahm and Rob Burton for performing all the opsonophagocytic assays so efficiently. Centre for International Child Health I am grateful to Prof Trevor Duke for allowing me to stay on in Fiji to continue my writing, keeping me funded, and allowing for me to get involved in other activities whilst writing up my thesis. I am grateful to Amanda O Brien for her groundbreaking perseverance in overcoming the many administrative challenges and to Amy Auge, Kathryn Gilbert, Evan Willis, Eleanor Neal, Joelle Milne, and Caitlyn Henry for all their help over the years. I am grateful to Dr Catherine Satzke who took charge of a large number of specimens, organised the serotyping, sorted out the many technical issues surrounding the microbiology, and agreed to inherit and finish a number of outstanding microbiology manuscripts. I am very thankful to Dr Adam Jenney who innocently agreed to do my job as on-site PI whilst I went on maternity leave to enjoy my family. I was grateful to be able to hand over the project to his capable hands (during a coup) and that he was able to continue to lead some aspects of our work on my return. In addition, I am extremely grateful that he offered (without duress) to read my thesis, correct my poor grammar, and help structure many aspects of it. Clinical Epidemiology and Biostatistics Unit I wish to thank Philip Greenwood for designing the database and providing training, supervision, and ongoing support for our many databases. Thanks to Suzanna Vidmar for devising the randomisation lists and envelopes and answering other statistical questions.

13 xii Royal Children s Hospital I am grateful to Chris Pearce who came to Fiji, assessed the microbiology lab, wrote many of the microbiology SOPs, helped with our supplies list, and answered my silly questions. I thank Gena Gonis for doing our sensitivity testing QC free of charge. Others I am very thankful for the many people who helped with many technical aspects of the project: Lorraine Kelpie for her expertise in performing venipuncture with a smile, Dr Vanessa Johnston, Dr Loretta Thorn, and Elizabeth Hamilton. I wish to thank Dr Graham Byrnes for his statistical input at the trial s inception and re-design stage. I am thankful to Prof Yin Bun Cheung who made himself available to provide oversight of the statistical analysis, his incredible ease and efficiency at sorting out my analytical problems, and explaining logistic regression in a language that I could understand. I thank Shirley Warren for providing training in microbiology and serotyping, and getting involved in the many aspects of the lab particularly in helping the lab identify resistant organisms during a nosocomial outbreak. I wish to thank Prof Porter Anderson, Prof Brian Greenwood, Dr George Siber, and Dr David Klein who had inputs to the study design at various stages. I wish to thank the members of the Data Safety and Monitoring Board whose guidance, support, and expertise were appreciated. I wish to thank CSL Biotherapies, Australia in particular Phillipe Ludekins, who arranged for the donation of Pneumovax TM. I thank GlaxoSmithKline who donated the coadministered Tritanrix TM -HepB TM and Hiberix TM vaccines and 2 vaccine fridges. NIH I am thankful to the staff at NIH particularly Elizabeth Horigan who we came to know when the study was being redesigned. Her expertise, encouragement, and support were always valuable. I wish to thank Farukh Khambaty who was always very supportive of our work and I am appreciative of all the assistance he has provided. Funding Bodies Funding was provided by NIAID (grant number R01 AI 52337) and the National Health and Medical Research Council. For 18 months the funding to write up my thesis was made possible by a National Health and Medical Research Council Public Health postgraduate scholarship.

14 xiii Supervisors I wish to thank Prof Jonathan Carapetis who has always been very positive, encouraging, supportive, and clear thinking, over the many years this work has developed and evolved. I have valued his advice, knowledge, and continued interest in the work. None of this work would have been possible without my supervisor Prof Kim Mulholland. I have learnt much from Kim over the years. His perseverance, when others would have given up, is extraordinary, and the belief in a better life for the world s children has always been forefront in his decision making. His encouragement and openness to get involved in other interesting research activities has been a blessing and a welcome distraction at times, and hence the long evolution of my thesis. My professional life has been enriched by the many opportunities that Kim has provided for me, for which I am truly grateful. Family and friends I am grateful to our nanny Ateca and the many Fijian nannies who welcomed my children into their hearts with love and grace, and created such a wonderful, memorable childhood for my children whilst I was at work and writing. For the support of my friends in Fiji who I could always call upon, and the delight we had in watching each others children grow. Most importantly, I would like to thank my husband, Alberto, who embarked on a Pacific journey, which was meant to be 2 years and lasted over 6 years. His patience, compromise, support, and understanding throughout times of chaos have been extraordinary and I am truly grateful to him. My two adorable children, Elian and Lucia, who think they are Fijian, and will miss Fiji and their close friends they have grown up with. I thank them for putting up with a working mother. Thank you and Vinaka Vakalevu Fiona Russell

15 xiv PUBLICATIONS 1. Russell FM, Mulholland EK. Recent advances in pneumococcal vaccination of children. Ann Trop Paediatr 2004;24(4): Magree HC, Russell FM, Sa'aga R, Greenwood P, Tikoduadua L, Pryor J, Waqatakirewa L, Carapetis JR, Mulholland EK. Chest X-ray-confirmed pneumonia in children in Fiji. Bull World Health Organ. 2005;83(6): Russell FM, Biribo S, Selvaraj G, Oppedisano F, Warren S, Seduadua A, Mulholland EK, Carapetis JR. Citrated sheep blood agar is a practical bacterial culture medium to replace citrated human blood agar in developing country laboratories. J Clin Microbiol 2006;44(9): Russell FM, Carapetis JR, Ketawai S, Kunabuli V, Taoi M, Biribo S, Seduadua A, Mulholland EK. Pneumococcal nasopharyngeal carriage and penicillin resistance patterns in young children in Fiji. Ann Trop Paediatr 2006;26(3): Russell FM, Balloch A, Tang MLK, Carapetis JR, Licciardi P, Nelson J, Jenney AWJ, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Cheung YB, Mulholland EK. Immunogenicity Following One, Two, or Three Doses of the 7-valent Pneumococcal Conjugate Vaccine. Vaccine 2009;27(41): Jin P, Kong F, Xiao M, Oftadeh S, Zhou F, Liu C, Russell F, Gilbert GL. First report of putative Streptococcus pneumoniae serotype 6D among nasopharyngeal isolates from Fijian children. J Infect Dis 2009;200(9): Russell FM, Carapetis JR, Balloch A, Licciardi PV, Jenney AWJ, Tikoduadua L, Waqatakirewa L, Pryor J, Nelson J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Hyporesponsiveness to Rechallenge Dose Following Pneumococcal Polysaccharide Vaccine at 12 Months of Age in a Randomised Controlled Trial. Vaccine 2010;28(19): Russell FM, Carapetis JR, Satzke C, Tikoduadua L, Waqatakirewa L, Chandra R, Seduadua A, Oftadeh S, Cheung YB, Gilbert GL, Mulholland EK. Pneumococcal Nasopharyngeal Carriage Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Schedule in Infancy and a 23-valent Pneumococcal Polysaccharide Vaccine Booster, a Randomized Controlled Trial. Accepted Clin Vaccine Immun Russell FM, Carapetis JR, Tikoduadua L, Chandra R, Seduadua A, Satzke C, Pryor J, Buadromo E,

16 xv Waqatakirewa L, Mulholland EK. Invasive Pneumococcal Disease in Fiji: clinical syndromes, epidemiology, and the potential impact of pneumococcal conjugate vaccine. Pediatr Infect Dis J Sep;29(9): Satzke C, Seduadua A, Carapetis JR, Chandra, Mulholland EK, Russell FM. Comparison of citrated human, citrated sheep and defibrinated sheep blood Mueller Hinton agar for antimicrobial sensitivity testing of Streptococcus pneumoniae isolates. J Clin Microbiol Jul Balloch A, Licciardi P, Russell FM, Burton R, Lin J, Nahm M, Mulholland EK, Tang MLK. 23-valent pneumococcal polysaccahruide is immunogenic in children at one year of age. J Allergy Clin Immunol Aug;126(2): Russell FM, Licciardi PV, Balloch A, Biaukula V, Tikoduadua L, Carapetis JR, Nelson J, Jenney AWJ, Waqatakirewa L, Colquhoun S, Cheung YB, Tang MLK, Mulholland EK. Safety and Immunogenicity of the 23-Valent Pneumococcal Polysaccharide Vaccine at 12 months of age, following One, Two, or Threes Doses of the 7-valent Pneumococcal Conjugate Vaccine in Infancy. Vaccine (18): Licciardi PV, Balloch A, Russell FM, Mulholland EK, Tang ML. Antibodies to serotype 9V exhibit novel serogroup cross-reactivity following infant pneumococcal immunization. Vaccine 2010;28(22): Satzke C, Ortika BD, Oftadeh S, Russell FM, Robins-Browne R, Mulholland EK, Gilbert GL. Molecular epidemiology of Streptococcus pneumoniae serogroup 6 isolates from Fijian children, including newly identified serotypes 6C and 6D. J Clin Microbiol Sep 1.

17 xvi POSTERS AND CONFERENCE PRESENTATIONS ORAL PRESENTATIONS Public Health Association of Australia Conference on Immunisation, August 2006 (1) Russell FM, Carapetis JR, Colquhoun S, Kunabuli V, Magree H, Seduadua A, Pryor J, Tikoduadua L, Waqatakirewa L, Mulholland EK. High ethnic disparity in invasive pneumococcal disease and pneumonia in Fiji. (2) Russell FM, Carapetis JR, Tang M, Balloch A, Colquhoun S, Nelson J, Pryor J, Tikoduadua L, Waqatakirewa L, Byrnes G, Mulholland EK. Immunogenicity following 1-3 doses of the 7-valent pneumococcal conjugate vaccine followed by the 23-valent pneumococcal polysaccharide vaccine booster. NIH DMID international investigator s meeting, Bethesda, USA, May 2007 (1) Russell F, Carapetis J, Tang M, Balloch A, Nelson J, Jenney A, Waqatakirewa L, Pryor J, Tikoduadua L, Byrnes G, Mulholland EK Pneumococcal opsonophagocytic assay results following a primary series of 0-3 doses of pneumococcal conjugate vaccine in infancy followed by a 12 month booster and 17 month microdose of the 23-valent pneumococcal polysaccharide vaccine. Fiji Medical Association Conference, Suva, Fiji, September 2007 (1) Russell, FM. Pneumococcal Disease and Treatment Costs (2) Russell, FM. Penicillin Resistance Surveillance (3) Russell, FM. FiPP update 6th International Symposium on Pneumococci and Pneumococcal Diseases, Reykajek, June 2008 (1) Mulholland EK, Russell FM. Hyporesponsiveness and pneumococcal polysaccharide vaccine in

18 xvii Fijian infants. Public Health Association of Australia Conference on Immunisation, September 2008 (1) Russell FM, Tang MLK, Balloch A, Licciardi P, Carapetis JR, Nelson J, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Immunogenicity Following One, Two, or Three Doses of the 7-valent Pneumococcal Conjugate Vaccine. (2) Russell FM, Tang MLK, Balloch A, Licciardi P, Carapetis JR, Nelson J, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Booster response of Pneumovax at 12 months of age following one, two, or three doses of Prevenar and impact on carriage. 7th International Symposium on Pneumococci and Pneumococcal Diseases, Tel Aviv, March 2010 (1) Russell FM, Carapetis JR, Balloch A, Licciardi PV, Jenney AWJ, Tikoduadua L, Waqatakirewa L, Pryor J, Nelson J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Hyporesponsiveness to Rechallenge Dose Following Pneumococcal Polysaccharide Vaccine at 12 Months of Age. (2) Russell FM, Carapetis JR, Burton RL, Lin J, Licciardi PV, Balloch A, Tikoduadua L, Waqatakirewa L, Cheung YB, Tang MLK, Nahm MH, Mulholland EK. Opsonophagocytic Activity Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Infant Primary Series and 23-valent Pneumococcal Polysaccharide Vaccine at 12 Months of Age. Australasian Society of Infectious Diseases, Darwin, May 2010 Mulholland K, Russell FM. Hyporesponsiveness to re-challenge dose following pneumococcal polysaccharide vaccine at 12 months of age, a randomized controlled trial. Victorian Infection and Immunity Network student symposium. Walter and Eliza Institute, Melbourne, June Russell, FM. Hyporesponsiveness to re-challenge dose following pneumococcal polysaccharide

19 xviii vaccine at 12 months of age, a randomized controlled trial. Public Health Association of Australia Conference on Immunisation, August 2010 (1) Russell FM, Carapetis JR, Balloch A, Licciardi PV, Jenney AWJ, Tikoduadua L, Waqatakirewa L, Pryor J, Nelson J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Hyporesponsiveness to Rechallenge Dose Following Pneumococcal Polysaccharide Vaccine at 12 Months of Age in a Randomised Controlled Trial. (2) Russell FM, Carapetis JR, Satzke C, Tikoduadua L, Waqatakirewa L, Chandra R, Seduadua A, Oftadeh S, Cheung YB, Gilbert GL, Mulholland EK. Pneumococcal Nasopharyngeal Carriage Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Schedule in Infancy and a 23- valent Pneumococcal Polysaccharide Vaccine Booster, a Randomized Controlled Trial. POSTER PRESENTATIONS 9 th Annual Conference on Vaccine Research, Baltimore, USA, May 2006 (1) Kunabuli VL, Mulholland EK, Tikoduadua L, Seduadua A, Pryor J, Russell FM. Prospective meningitis burden of disease study and rapid assessment of neurological outcomes in children in Fiji. 5th International Symposium on Pneumococci and Pneumococcal Diseases, Alice Springs, April 2006 (1) Russell FM, Carapetis JR, Tang M, Balloch A, Colquhoun S, Nelson J, Pryor J, Tikoduadua L, Waqatikirewa L, Byrnes G, Mulholland EK. Immunogenicity following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine and booster response of the 23-valent pneumococcal polysaccharide vaccine at 12 months of age. (2) Russell FM, Biribo S, Selvaraj G, Oppedisano F, Warren S, Seduadua A, Mulholland EK, Carapetis JR. Comparison of citrated sheep and human blood with defibrinated horse and sheep blood as culture media supplements for the isolation and antibiotic susceptibility testing of Streptococcus

20 xix pneumoniae. (3) Russell FM, Carapetis JR, Ketawai S, Kunabuli V, Taoi M, Biribo S, Seduadua A, Mulholland EK. Pneumococcal Nasopharyngeal Carriage and Penicillin Resistance Patterns in Young Children in Fiji. (4) Kunabuli VL, Mulholland EK, Tikoduadua L, Seduadua A, Pryor J, Russell FM. Prospective meningitis burden of disease study and rapid assessment of neurological outcomes in children in Fiji. (5) Seduadua AN, Mulholland EK, Carapetis JR, Buadromo E, Russell FM. Comparison of antimicrobial sensitivity results on citrated sheep blood Mueller Hinton and citrated human blood Mueller Hinton for invasive Streptococcus pneumoniae clinical isolates. (6) Biribo S, Russell FM, Carapetis JR, Kataiwai S, Mulholland EK. Multiserotype nasopharyngeal carriage of Streptococcus pneumoniae in infants in Fiji. (7) Colquhoun SM, Russell FM, Carapetis JR, Tikoduadua LV, Pryor J, Waqatakirewa L, Mulholland EK. Ethnic disparity in the burden of invasive pneumococcal disease in children aged less than 5 years in Fiji. (8) Colquhoun SM, Russell FM, Carapetis JR, Tikoduadua L, Pryor J, Wake M, Mulholland, EK. A cohort study to assess quality of life in young Fijian children who have a history of bacterial meningitis. 6th International Symposium on Pneumococci and Pneumococcal Diseases, Reykajek, June 2008 (1) Russell FM, Tang MLK, Balloch A, Licciardi P, Carapetis JR, Nelson J, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Immunogenicity Following One, Two, or Three Doses of the 7-valent Pneumococcal Conjugate Vaccine. (2) Russell FM, Tang MLK, Balloch A, Licciardi P, Carapetis JR, Nelson J, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Booster response of Pneumovax at 12 months of age following one, two, or three doses of Prevenar. (3) Russell FM, Carapetis JR, Chandra R, Tikoduadua L, Waqatakirewa L, Seduadua A, Pryor J, Satzke C, Gosling D, Mulholland EK. Nasopharyngeal Carriage of Pneumococcal Vaccine Types Following 0, 1, 2, or 3 doses of 7-valent Pneumococcal Conjugate Vaccine.

21 xx (4) Russell FM, Carapetis JR, Kunabuli V, Nelson J, Jenney A, Bright K, Tang MLK, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Safety of the 23-valent pneumococcal polysaccharide vaccine at 12 months of age following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine. (5) Russell FM, Chandra R, Carapetis JR, Seduadua A, Tikoduadua L, Buadromo E, Waqatakirewa L, Pryor J, Mulholland EK. Epidemiology and Serotypes of Invasive Pneumococcal Disease in all ages in Fiji. (6) Russell FM, Carapetis JR, Tikoduadua L, Waqatakirewa L, Pryor J, Mulholland EK. Estimated Impact of the 7-valent Pneumococcal Conjugate Vaccine in Fiji. (7) Kunabuli V, Mulholland EK, Tikoduadua LV, Azzopardi K, Robins-Browne R, Wake M, Seduadua A, Chandra R, Richmond P, Pryor J, Russell FM. Aetiology and Outcomes of Meningitis in Children in Fiji. (8) Chandra R, Mulholland K, Buadromo E, Seduadua A, Carapetis JR, Russell FM. Pneumococcal antimicrobial resistance patterns in clinical invasive isolates in Fiji. (9) Russell FM, Kunabuli V, Griffiths UK, Tikoduadua L, Carapetis JR, Waqatakirewa L, Pryor J, Magree H, Mulholland EK. Cost of Pneumococcal Disease in Fiji. (10) Temple B, Tikoduadua LV, Mulholland EK, Griffiths UK, Russell FM. Cost of Outpatient Pneumonia in Children Less than 5yrs of Age in Fiji. (11) Balloch A, Licciardi PV, Russell FM, Mulholland EK, Tang MLK. The ability of infants to respond to 23-valent unconjugated Pneumovax at 12 months in the absence of prior 7-valent conjugated Prevenar immunisation. (12) Balloch A, Licciardi PV, Russell FM, Burton R, Nahm M, Mulholland EK, Tang MLK. Does serotype specific IgG and avidity to Streptococcus pneumoniae following infant immunisation correlate to functional opsonophagocytic activity? (13) Licciardi PV, Balloch A, Russell FM, Mulholland EK, Tang MLK. Cross reactive antipneumococcal antibodies.

22 xxi Public Health Association of Australia Conference on Immunisation, September 2008 (1) Russell FM, Chandra R, Carapetis JR, Seduadua A, Tikoduadua L, Buadromo E, Waqatakirewa L, Pryor J, Mulholland EK. Epidemiology and Serotypes of Invasive Pneumococcal Disease in all ages in Fiji. Physicians Week 2009, Sydney (accepted but withdrawn as unable to attend) 1) Russell FM, Balloch A, Biaukula V, Tikoduadua L, Carapetis JR, Licciardi P, Nelson J, Jenney AWJ, Waqatakirewa L, Pryor J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Immunogenicity Following Reduced Doses of Pneumococcal Conjugate Vaccine and 12 month Pneumococcal Polysaccharide Vaccine Booster. 2) Russell FM, Carapetis JR, Balloch A, Licciardi P, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Nelson J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Hyporesponsiveness to challenge dose following pneumococcal polysaccharide at 12 months of age. 7th International Symposium on Pneumococci and Pneumococcal Diseases, Tel Aviv, March 2010: (1) Russell FM, Balloch A, Licciardi PV, Carapetis JR, Tikoduadua L, Waqatakirewa L, Cheung YB, Mulholland EK, Tang MLK. Serotype-specific avidity is achieved following a single dose of the 7- valent pneumococcal conjugate vaccine, and is enhanced by 23-valent pneumococcal polysaccharide booster at 12 months. (2) Russell FM, Carapetis JR, Satzke C, Tikoduadua L, Waqatakirewa L, Chandra R, Seduadua A, Oftadeh S, Cheung YB, Gilbert GL, Mulholland EK. Pneumococcal Nasopharyngeal Carriage Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Schedule in Infancy and a 23- valent Pneumococcal Polysaccharide Vaccine Booster, a Randomized Controlled Trial.

23 xxii AWARDS I was fortunate to receive the following awards/fellowships during the course of my PhD: Quintiles Fellowship, Royal Australasian College of Physicians, 2007 in which I undertook a 10 day Advanced Course in Epidemiological Analysis at the London School of Hygiene and Tropical Medicine. National Health and Medical Research Council Scholarship, to write up my PhD. Early in Career Public Health Award in Immunisation, Public Health Association of Australia, 2008 for the presentation: Booster response of Pneumovax at 12 months of age following one, two, or three doses of Prevenar and impact on carriage at the Public Health Association of Australia Conference on Immunisation, September 2008.

24 xxiii TABLE OF CONTENTS 1 LITERATURE REVIEW Background Information Pneumococcal Disease Bacteriology Pathogenesis Pneumococcal Disease Burden Global Pneumococcal Serotype Distribution Pneumococcal Pneumonia Burden of Pneumococcal Disease in Fiji Protection Against Pneumococcal Disease Host Defense Mechanisms Pneumococcal Vaccines Rationale Access to Vaccines Evaluation of Alternative Pneumococcal Vaccine Schedules is a Research Priority Immunological Basis to the Vaccine Trial Design Knowledge to be Gained Evaluation of Alternative Pneumococcal Schedules in Fiji Objectives Change to the Original Objectives Potential Risks Known Potential Benefits Hypothesis MATERIALS AND METHODS Setting Health Infrastructure Drug Licensing Procedure Study Sites Suva Study Team Ethical Procedures Study Design Selection of Study Participants Informed Consent Eligibility Criteria Enrollment, Randomisation and Masking Procedures Study Vaccines, Vaccine Storage and Administration Study Visits... 58

25 xxiv Follow-up Withdrawal of a Participant From the Study Assessment of Safety Sample Collection Sample Transportation - International Antibody Assays Data Management Data capture methods Clinical Monitoring Plan Source Documents Protocol Deviations Quality Control and Quality Assurance Statistical Methods Background to Original Study Protocol Sample Size Calculation Definition of Outcome Measures Statistical Analysis of Primary Objective Statistical Analysis of Secondary Objectives Statistical Analysis of Tertiary Objectives PNEUMOCOCCAL NASOPHARYNGEAL CARRIAGE AND PATTERNS OF PENICILLIN RESISTANCE IN YOUNG CHILDREN IN FIJI Abstract Introduction Methods Study Site Study Design Risk Factor Evaluation Laboratory Methods Statistical Analysis Ethics Approval Results Discussion EPIDEMIOLOGY OF INVASIVE PNEUMOCOCCAL DISEASE IN FIJI: THE POTENTIAL IMPACT OF PNEUMOCOCCAL CONJUGATE VACCINE Abstract Introduction Methods Results... 96

26 xxv 4.5 Discussion IMMUNOGENICITY FOLLOWING ONE, TWO, OR THREE DOSES OF THE 7- VALENT PNEUMOCOCCAL CONJUGATE VACCINE Abstract Introduction Methods Study Participants Study Procedures and Vaccines Laboratory Procedures Statistical Analysis Results Discussion SAFETY AND IMMUNOGENICITY OF THE 23-VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE, FOLLOWING ONE, TWO, OR THREES DOSES OF THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE IN INFANCY Abstract Introduction Methods Study Participants Study Procedures and Vaccines Laboratory Procedures Statistical Analysis Results Immunogenicity to PCV Serotypes Immunogenicity to Non-PCV Serotypes Adverse Events Discussion HYPORESPONSIVENESS TO RE-CHALLENGE DOSE FOLLOWING PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE, A RANDOMIZED CONTROLLED TRIAL Abstract Introduction Methods Study Participants Study Procedures and Vaccines Laboratory Procedures

27 xxvi Data Management and Statistical Analysis Ethical Approval Results Discussion OPSONOPHAGOCYTIC ACTIVITY FOLLOWING A REDUCED DOSE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE INFANT PRIMARY SERIES AND 23- VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE Abstract Introduction Materials and Methods Study Participants Study Procedures and Vaccines Laboratory Assays Statistical Analysis Results PCV Serotypes Non-PCV and PCV related serotypes Discussion SEROTYPE-SPECIFIC AVIDITY IS ACHIEVED FOLLOWING A SINGLE DOSE OF THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE, AND IS ENHANCED BY 23- VALENT PNEUMOCOCCAL POLYSACCHARIDE BOOSTER AT 12 MONTHS Abstract Introduction Methods Study Participants Study Procedures and Vaccines Laboratory Procedures Statistical Analysis Results Discussion PNEUMOCOCCAL NASOPHARYNGEAL CARRIAGE FOLLOWING REDUCED DOSES OF 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE AND A 23-VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE BOOSTER Abstract Introduction Methods

28 xxvii Study Design Nasopharyngeal Swabs Questionnaires Statistical Analysis Results Discussion CONCLUSIONS Implications for Pneumococcal Vaccine Policy in Fiji and Other Countries REFERENCES APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4

29 xxviii LIST OF TABLES Table 1: Table 2: Summary of serotype-specific GMC data from trials of CRM197-conjugated pneumococcal vaccines, one month post primary series Summary of serotype-specific GMC data from trials of reduced dose pneumococcal conjugate vaccines Table 3: Outline of study visits Table 4: Characteristics of the study children (n=440) Table5: Table 6: Table7: Antimicrobial resistance patterns of Streptococcus pneumoniae isolates (n=246) Level of antimicrobial resistance of Streptococcus pneumoniae isolates (n=246) Serogroups/types of Streptococcus pneumoniae isolates from study children (n=239) Table8: Risk factors for pneumococcal nasopharyngeal carriage Table 9: Clinical manifestations of IPD cases by age (n=83) Table 10: Table 11: Table 12: Estimated annual number of IPD and hospitalized chest X-ray confirmed pneumonia cases and deaths in <5 year olds in Fiji, and the estimated number of cases and deaths averted if PCV were introduced Baseline characteristics of infants at enrolment and randomised to the different PCV groups Geometric mean concentrations (GMC) of serotype-specific IgG titres taken 4 weeks following the PCV primary series, and at 9 and 12 months of age, by number of PCV doses administered in the primary series Table 13: Proportion of infants with antibody concentrations 0.35 and 1μg/mL at 4 weeks post primary series, and at 9 and 12 months of age, by number of PCV doses administered in the primary series Table 14: Serotype-specific IgG geometric mean concentrations (GMC and 95% confidence intervals) to PCV serotypes before and 14 days following the 12 month 23vPPS and by number of PCV doses administered in the primary series Table 15: Table 16: Proportions of children with antibody concentrations 0.35 and 1µg/mL to PCV serotypes before and 14 days post-12 month 23vPPS and by number of PCV doses administered in the primary series Serotype-specific IgG GMC (and 95% confidence intervals) and proportions of children with antibody concentrations 0.35 and 1µg/mL to PCV serotypes at 17 months in those who did or did not receive the 12 month 23vPPS and by number of PCV doses in the primary series...127

30 xxix Table 17: Table 18: Serotype-specific IgG GMC (and 95%CI) and proportions of children with antibody concentrations 0.35 and 1µg/mL to non-pcv serotypes before and 14 days post-12 month 23vPPS Serotype-specific IgG GMC (and 95% confidence intervals) and proportions of children with antibody concentrations 0.35 and 1µg/mL to non-pcv serotypes at 17 months of age in those that did or did not receive the 12 month 23vPPS Table 19: Non-serious adverse events1 in those children who received 23vPPS at 12 months of age (n=245) Table 20: Table 21: Table 22: Table23: Table 24: Table 25: Baseline characteristics of infants at enrolment and on randomisation to one of eight groups Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of infants with OI 8 to 6 PCV serotypes 4 weeks post primary series, and at 9 and 12 months of age, by number of PCV administered in the primary series OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI 8 to 6 PCV serotypes pre- and 14 days post-23vpps at 12 months of age and by number of PCV administered in the primary series Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI)1, OPA Geometric Mean Titers2 (GMT and 95%CI) and proportions of infants with OI 8 pre-mpps at 17 months of age and one month post-mpps in those that have or have not received the 12 month 23VPPS and by number of PCV doses in the primary series Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI 8 to the non-pcv serotypes 1 and 5 pre- and 14 days post-23vpps at 12 months of age Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI 8 to 7 non-pcv serotypes at 17 months and one month post-mpps Table 26: Timing of vaccination and blood draws for each of the 8 groups Table27: Table 28: Table29: Geometric mean (GM) concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of serotype-specific that is avid) for the PCV serotypes, taken 4 weeks following the PCV primary series, and at 9 and 12 months of age Geometric mean concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of serotype-specific IgG that is avid (1) to PCV serotypes before and 14 days post 12 month 23vPPS, by number of PCV doses administered in the primary series in children randomized to receive 12 month 23vPPS Geometric mean concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of antibody that is avid) to PCV serotypes pre-mpps at

31 xxx Table30: Table31: 17 months and one month post-mpps in those who did or did not receive the 12 month 23vPPS shown by number of PCV doses in the primary series Geometric mean concentrations of serotype-specific IgG and median AI (MAI1, percentage of antibody that is avid) to non-pcv serotypes before and 14 days post 12 month 23vPPS (n=218) in infants randomized to receive 12 month 23vPPS Geometric mean concentrations of serotype-specific IgG and median AI (MAI1, percentage of serotype specific IgG that is avid) to non-pcv serotypes at 17 months of age and one month post mpps, in those that did or did not receive the 12 month 23vPPS Table32: Timing of vaccination and blood draws for each of the 8 groups Table 33 Table 34: Table 35: Characteristics of infants by group allocation at enrolment and at each of the 4 nasopharyngeal swab visits (%, unless otherwise stated) Nasopharyngeal (NP) carriage of all pneumococcal, 7-valent pneumococcal conjugate vaccine (PCV) serotypes (VT), and non-pcv serotypes (NVT) at 6, 9, and 12 months (m) of age following administration of 0, 1, 2, or 3 doses of PCV as a primary series Nasopharyngeal (NP) carriage of all pneumococcal and non-pcv serotypes (NVT) at 17 months of age in those who did or did not receive the 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months of age

32 xxxi LIST OF FIGURES Figure 1: The annual incidence of hospitalised pneumonia (ICD10: J12-18) for all ages, for Viti Levu Figure 2: Map of the Republic of the Fiji islands showing the 4 medical divisions Figure 3 : Figure 4: Flowchart of nasopharyngeal swabs and sensitivity and serotype of pneumococcal isolates Annual incidence of IPD by age group in the Central Medical Division, Fiji from 1st July 2004 to 31st October, Figure 5: IPD case fatality rates in the Central Medical Division, Fiji, by age group Figure 6: Serotype distribution amongst IPD cases (n=78) Figure 7: Figure 8: Figure 9: Figure 10: Figure 11: Figure 12: Figure 13: Figure 14: Proportion of IPD isolates, by age, potentially covered by the 7, 10, and 13-valent pneumococcal conjugate vaccine, and the 23-valent pneumococcal polysaccharide vaccine CONSORT chart of the screened and enrolled children to 12 months of age CONSORT chart of the screened and enrolled children to 17 months of age CONSORT chart of the screened and enrolled children to 18 months of age, showing the number having the pre-mpps blood test1, mpps at 17 months of age2, and blood test one month post-mpps Serotype-specific IgG GMC (μg/ml) to PCV serotypes at 17 months of age pre-mpps Serotype-specific IgG GMC (μg/ml) to PCV serotypes at 17 months of age post-mpps in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs Serotype-specific IgG GMC (μg/ml) to non-pcv serotypes at 17 months of age pre-mpps in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs Serotype-specific IgG GMC (μg/ml) to non-pcv serotypes one month post-mpps in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs

33 xxxii Figure 15: Figure 16: Figure 17: Figure 18: Figure 19: Figure 20: Figure 21: Figure 22: Figure 23: Figure 24: Figure 25: Pre- and one month post-mpps log antibody concentrations for non-pcv serotypes 1, 5, 7F, and 19A in those that did (+) and did not (o) receive 23vPPS at 12 months of age. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs Pre- and one month post-mpps log antibody concentrations for serotype 4 for children who did (+) or did not (o) receive 23vPPS at 12 months of age Pre- and one month post-mpps log antibody concentrations for serotype 6B for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series Pre- and one month post-mpps log antibody concentrations for serotype 4 for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series Pre- and one month post-mpps log antibody concentrations for serotype 6B for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series Pre- and one month post-mpps OPA titer for serotypes 4 and 6B, in those that did (+) and did not (o) receive 23VPPS at 12 months of age. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs Median AI (MAI, percentage of serotype-specific-igg that is avid) for the PCV serotypes taken 4 weeks following the PCV primary series, and at 9 and 12 months of age Median percentage of serotype-specific IgG that is avid for the PCV serotypes pre and post 12 month 23vPPS and pre and post 18 month mpps in those that did or did not receive the 12 month 23vPPS Median AI (percentage of serotype-specific IgG that is avid) for selected non-pcv serotypes in those that did or did not receive the 12 month 23vPPS Cumulative proportion of infants carrying a 7-valent pneumococcal conjugate vaccine (PCV) type at 6, 9, 12, and 17 months of age by PCV and 23-valent pneumococcal polysaccharide vaccine (23vPPS) group allocation Nasopharyngeal (NP) carriage rates of all pneumococcal and 7-valent pneumococcal conjugate vaccine (PCV) serotypes (VT) at 17 months of age following 0, 1, 2, or 3 PCV doses of PCV in infancy with or without 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months

34 xxxiii ABBREVIATIONS AE AI ARI CFR CI C-PS CRF CRP CSF CWMH CXR DMID DSMB ELISA FiPP FNRC GAVI GCP GMC GMT Hib HIV ICD ICH Ig ILN IMR IPD IQR Adverse event Avidity index Acute respiratory infection Case fatality rate Confidence interval Cell wall polysaccharide Case reporting form C-reactive protein Cerebrospinal fluid Colonial War Memorial Hospital Chest X-ray Department of Microbiology and Infectious Diseases Data safety and monitoring board Enzyme-linked immunosorbent assay Fiji pneumococcal project Fiji National Research Committee Global Alliance for Vaccines and Immunisation Good clinical practice Geometric mean concentration Geometric mean titre Haemophilus influenzae type b Human immunodeficiency virus International classification of diseases International Conference on Harmonisation Immunoglobulin Interleukin Infant mortality rate Invasive pneumococcal disease Interquartile range

35 xxxiv IRR IU LRTI MCH MoH NHMRC NIAID NIH NK NP NVT OI OPA PCV PedsQL PneumoADIP PI PPS RR SAE TLR TNF UK US VT WC WHO 23vPPS Incidence rate ratio International units Lower respiratory tract infection Maternal and Child Health Ministry of Health National Health and Medical Research Council National Institute of Allergy and Infectious Diseases National Institutes of Health Natural killer Nasopharyngeal Pneumococcal non-vaccine type Opsonization index Opsonophagocytic activity 7-valent pneumococcal conjugate vaccine Pediatric quality of life questionnaire Pneumococcal Vaccines Accelerated Development and Introduction Plan Principal investigator Pneumococcal polysaccharide vaccine Relative risk Serious adverse event Toll like receptor Tumour necrosis factor United Kingdom United States of America Pneumococcal vaccine type White cell count World Health Organization 23-valent pneumococcal polysaccharide vaccine

36 1 1 LITERATURE REVIEW 1.1 Background Information Streptococcus pneumoniae (pneumococcus) was first identified by Sternberg in the US [1] and Pasteur in France in 1880 [2]. It is the most common cause of bacterial pneumonia in children worldwide and is the leading vaccine preventable cause of serious infection in infants [3]. An estimated 1.6 million deaths are attributable to pneumococcal disease each year with the majority of these deaths occurring in low income countries primarily in children and the elderly [4]. The case fatality rate (CFR) is particularly high in infants <6 months old [5]. Vaccines to prevent pneumococcal disease were first developed after the recognition of the high morbidity and mortality in South African gold miners in the early 20 th century [6]. Several trials of polyvalent pneumococcal vaccines were undertaken. However the choice of controls potentially leading to bias and overestimating the vaccine efficacy, and the lack of accompanying bacteriology cast doubt over the findings [7]. The introduction of sulphonamide antibiotics in the late 1930s put a hold on further vaccine development. However, more recently, antibiotic resistance and the growing awareness of the pneumococcal burden, had become significant issues in many parts of the world necessitating the development of effective vaccines to prevent pneumococcal disease [8]. 1.2 Pneumococcal Disease S.pneumoniae colonises the mucosal surfaces of the human nasopharynx and upper airway. Through a combination of virulence-factor activity and an ability to evade the early components of the innate host immune response, S.pneumoniae can spread locally to the middle ear to cause acute otitis media (OM), to the paranasal sinuses to cause sinusitis, or can spread from the upper respiratory tract to the sterile regions of the lower respiratory tract, resulting in pneumonia. In addition, pneumococci may cause systemic infections often associated with considerable mortality, including bacteraemia and meningitis, or in rare cases, septic arthritis, peritonitis, osteomyelitis, and soft tissue infections. Invasive pneumococcal disease (IPD) refers to any pneumococcal infection occurring in a normally sterile site. Bacteraemic infection occurs as a complication of either pneumonia or direct spread from the pharynx. Thus all pneumococcal disease commences with colonisation [9-11]. Host and bacterial factors contribute to IPD pathogenicity. Ethnicity, extremes of age,

37 2 co-morbidities, alcoholism, and immunosuppression including HIV are well known risk factors of IPD [12, 13]. Secretions from colonised individuals are thought to be responsible for person-to-person spread of the organism. An individual strain can be carried for weeks to months before its eventual clearance. Colonisation is most common in early childhood and acquisition of one or more strains occurs sequentially or simultaneously [14]. Nasopharyngeal (NP) carriage rates vary by age, ethnicity, and geographical location. In developing countries, NP colonisation rates can be >60% by 2 months of age [15, 16]. In addition the rate of NP carriage by capsular serotype varies. The factors that influence these differences are not well understood. The importance of the reduction or prevention of NP carriage in children on the spread of the organism has recently been demonstrated in the US whereby the widespread use of infant 7-valent pneumococcal conjugate vaccine (PCV, Prevenar, Pfizer Inc.) resulted in substantial indirect effects [17-19]. Indirect benefits of PCV exceeded the direct effects, with more than twice the number of cases of vaccine type (VT) IPD prevented in unvaccinated persons compared with the number of cases prevented in vaccinated children [19]. The reduction in pneumococcal VT carriage in children interrupted the transmission of pneumococci to close unvaccinated contacts [17, 18]. 1.3 Bacteriology S.pneumoniae is a Gram-positive diplococcus. It is a fastidious, facultative anaerobic organism that grows in short chains. Serotypes 3 and 37 grow as large mucoid colonies but other serotypes produce smooth colonies. Pneumococci lack catalase or peroxidase. As hydrogen peroxide is one of the end products of pnumococci s metabolism, by adding red blood cells to the growth medium the inactivation of hydrogen peroxide enhances pneumococci s viability. S.pneumoniae alters haemoglobin under aerobic conditions, producing a greenish discolouration of the surrounding blood-containing medium. The sensitivity of pneumococci to ethylhydrocupreine (Optochin) is widely used for laboratory diagnosis. However some isolates are Optochin resistant [20] and bile solubility is usually regarded as definitive for routine diagnostic purposes. Culture of pneumococcus from a normally sterile site still remains the most specific diagnostic gold standard. Despite the high specificity of bacterial culture, the diagnosis of pneumococcal infection from bacterial culture has numerous limitations. Prior treatment with antibiotics, delays in specimen transport to the laboratory, and other laboratory factors

38 3 such as the use of human blood agar to culture the organism may reduce isolation rates. Defibrinated sheep, horse, pig or goat blood agar is recommended for the isolation of S. pneumoniae [21-23]. Agar prepared using human blood is not recommended, partly because of the safety risk to laboratory personnel, but mainly because of poor bacterial isolation rates. A study evaluating the growth of S.pneumoniae on human blood agar compared with the gold standard horse or sheep blood agar found smaller colony size and absent or minimal hemolysis on human blood agar [24]. Despite this, it is common practice in many low income countries to prepare bacterial culture media using expired human blood obtained from donors for blood transfusions, because it is convenient and inexpensive [25]. In addition, most cases of pneumococcal pneumonia are non-bacteraemic so this test is less helpful for diagnosing the most common disease manifestation of this organism. 1.4 Pathogenesis S.pneumoniae has a plethora of virulence factors. An array of virulence factors needs to be expressed in a co-ordinated fashion for tissue invasion to be successful. Important virulence factors include the polysaccharide capsule, the cell wall, choline-binding proteins, pneumococcal surface proteins A and C, the LPTXG-anchored neuroaminidase proteins, hyaluronate lyase, pneumococcal adhesion and virulence A, enolase, pneumolysin, autolysin, and the metal-binding proteins pneumococcal surface antigen A, pneumococcal iron acquisition A, and pneumococcal iron uptake A [14]. Pneumococcal capsular polysaccharide is the most important virulence factor of S.pneumoniae by virtue of its anti-phagocytic properties [26]. In addition, the expression of a capsule allows access to the epithelial surface and prevents entrapment in nasal mucus [27]. Capsule production is vital for pneumococcal virulence and the thickness of the capsule in a particular strain and serotype is related to virulence [28]. The capsule physically protects the bacterium from antibodies and complement. The potential for pneumococci to become invasive seems to be related to the polysaccharide composition [29-31] and serotype/group has been shown to be independently associated with IPD severity in adults [32-35]. This is likely to reflect their relative ability to resist phagocytosis and differences in eliciting a humoral immune response. The pneumococcus secretes a protease that cleaves the hinge region of IgA1, the most abundant immunoglobulin expressed at the host site [36]. As a result, the organism is left coated with fragments lacking Fc domains and therefore is able to evade recognition by Fc receptors or complement. A switch in the expression of important virulence factors is required for the transition from NP colonisation to the development of

39 4 invasive disease. In addition to capsular expression, the thickness of the pneumococcal capsule is an important factor in the degree to which pneumococci are exposed to other important pneumococcal surface structures. Several studies have evaluated the relationship between pneumococcal serotypes (capsule) and IPD. A Swedish study of 494 adults with IPD showed that certain pneumococcal serotypes with low IPD potential behave as opportunistic pathogens causing disease in fragile persons, whereas serotypes 1 and 7F, known to have a high IPD potential, acted as primary pathogens, causing infections in previously healthy people [37]. A retrospective study of 464 IPD cases among adults in Denmark showed that after adjusting for other markers of severity, infection with serotype 3 was associated with a higher CFR (RR 2.54; 95%CI, ) whereas infection with serotype 1 was associated with a decreased CFR (RR 0.23; 95%CI, ) [32]. In a large study in the US of 5,579 adults aged 50 with IPD, serotypes 3, 11A, 19F, and 23F were found to be associated with significantly higher CFR than was serotype 14 [19]. A retrospective cohort study from the Netherlands including 1,075 hospitalised IPD cases found serotypes 3, 19F, 23A, 16F, 6B, 9N, and 18C were associated with increased CFR (group adjusted OR, 2.6; 95% CI, ) indicating that serotype was independently associated with IPD severity in adults [38]. A retrospective study in German children aged <16 years with hospitalised IPD found that serotype 7F accounted for a higher risk of severe and fatal outcomes than other serotypes [35]. In contrast, a prospective multi-site study of 796 IPD cases assessed the association of serotype and host related factors with disease severity and mortality after adjusting for age and the presence of underlying conditions [12]. The results found that host factors were more important than isolate serotype in determining the severity and outcome of IPD [12]. However the most comprehensive study to date is a population-based study from Denmark of 18,858 IPD cases for those 5 years. Serotypes 31, 11A, 35F, 17F, 3, 16F, 19F, 15B, and 10A were associated with a higher CFR compared to serotype 1 after adjusting for age, IPD focus, underlying conditions, and other potential confounders (adjusted OR 3, p<0.001) suggesting specific serotypes independently affect IPD mortality [34]. Molecular epidemiological analysis has demonstrated that clonal type (based on profiles of housekeeping genes) in addition to capsular type, influences the potential of S.pneumoniae to cause invasive disease [39]. In one study from the UK, multilocus sequence typing was performed on a number of carriage and IPD isolates and the odds ratio of the invasiveness of isolates was calculated. The findings suggested that capsular serotype may be more

40 5 important than genotype [29]. However this study did not adjust for age and the presence of underlying conditions. In a study from Sweden involving 273 IPD isolates (mainly from adults) and 246 NP carriage isolates, clones that belonged to the same serotypes but had different abilities to cause IPD were found. In addition, isolates belonging to the same clone had different capsules because of serotype switch, and were found to have the same disease potential. These findings suggest that there are other factors, apart from capsular polysaccharide, that may be important in the ability of pneumococci to cause invasive disease [31]. Cell wall polysaccharide (C-PS) is unique to S.pneumoniae and is present in all isolates. It reacts with C-reactive protein (CRP) and activates the alternative complement pathway. Antibody to C-PS does not protect against pneumococcal infection, although it can be detected in virtually all children and adults [40]. 1.5 Pneumococcal Disease Burden In 2005, the World Health Organization (WHO) estimated that 1.6 million people die of pneumococcal disease annually [4]. Children under 5 years of age account for 0.7 to 1 million of these deaths each year, with most from low income countries [4]. A recent review estimated over 14 million episodes of serious pneumococcal disease worldwide, with over 800,000 deaths in children <5 years [41]. The burden of pneumococcal disease in the elderly in low income countries is unknown. In industrialised countries, young children and the elderly have the highest burden of pneumococcal disease [4]. Comparing disease burden rates between geographical sites is difficult due to underreporting, differences in surveillance and reporting methods, antibiotic prescribing practices, and disparities in blood culture practice and laboratory techniques. In Western Europe, the reported IPD rates in Sweden and Spain were 4.2 and 56.2 per 100,000 children aged <5 prior to vaccine introduction respectively [42]. In the UK, the IPD incidence rate prevaccine introduction was 24.3 per 100,000 <5 year olds [43]. In the US, incidence rates prior to vaccine introduction were many times higher than in Western Europe which probably reflected different clinical practice with a greater number of blood cultures being drawn particularly from outpatients [44]. In New Zealand, the rate of IPD in <5 year olds was 54.2 per 100,000 [45]. In Chile, a middle income country, the incidence rate for IPD was 32 per 100,000 children aged <5 years [46]. In Africa, which is comprised of mostly low income countries with high infant mortality rates (IMR), the incidence of IPD in children <5 years was

41 6 estimated to be 111 to 436 per 100,000 [47]. Recently WHO and the PneumoADIP standardised case definitions and supported a co-ordinated multi-site surveillance project of pneumococcal disease in Asia and Africa [48]. Where IPD incidence rates were calculated, Vietnam a low income country with low IMR, the incidence rate was 48.7 per 100,000 children aged <5 years [49]. In rural Thailand, a middle income country with low IMR had an IPD rate of per 100,000 <5s [50]. Thus IPD is a significant burden to communities. Similar to <5 year old data, rates of IPD in adults vary by geographical region. In 1998 prior to PCV introduction, the national incidence of IPD in the elderly population in the US was estimated to be 60 per 100,000 but rates varied between different regions in the US primarily due to blood culture practices [51]. Since the national introduction of PCV into the US infant schedule the vaccine has had a larger than expected herd immunity effect on IPD in the unvaccinated elderly and other non-vaccinated age groups [19, 52]. The rates of IPD significantly declined in all age groups: by 32% in 20 to 39 year olds, by 8% in 40 to 64 year olds, and by 18% for those aged 65 years or more [52]. Disparate IPD rates between different ethnic groups living within the same geographical region have been described. In an Auckland-based study, the incidence of IPD among Pacific Island children was nearly four times, and that among Maori over twice the rate in other ethnic groups [53]. The incidence of IPD in Australian Indigenous children in the pre-vaccine era was 3.2 times the rate compared to non-indigenous Australian children [54]. Similarly in Israel, the IPD incidence was 2-fold higher in Bedouin children compared with Jewish children [55]. White Mountain Apache persons had an 8-fold greater risk of IPD than did the general US population pre-vaccine [56]. These different IPD rates in different ethnic groups may be related to genetic susceptibility, poorer living conditions, or other unknown factors Global Pneumococcal Serotype Distribution The pneumococcus has a capsule composed of polysaccharide which completely envelops the pneumococcal cells. Ninety-two different capsular types of pneumococci have been identified and form the basis of antigenic serotyping of the organism [57] including the newly identified 6D discovered from our Fiji isolates [58]. Within serogroups, serotypes cross-react immunologically, but only in some cases does this appear to translate into crossprotection (eg 6B with 6A but not 19F with 19A). The association of particular serotypes with disease varies according to age, geography, and clinical site.

42 7 Serotype distributions change with age with a narrower range of serotypes causing disease in young children compared to older children and adults [59, 60]. For children <5 years old, serotype 14 is the commonest serotype causing IPD worldwide, and serotype 1 and 5 are ranked in the top 3 serotypes in the Global Alliance for Vaccines and Immunisation (GAVI)- eligible low income countries [61]. In contrast, serotypes 18C, 4, and 9V are more common causes of IPD in North America and Oceania in <5 year olds than other regions of the world [61]. In all regions of the world, 3 serotypes (1, 5, 14) account for 28-43% of IPD in <5 year olds [61]. These are the same 3 serotypes found in lung aspirate study on pneumonia cases in the US in 1941 [62]. Seven serotypes (1, 5, 6A, 6B, 14, 19F, 23F) account for approximately 58-66% of all IPD worldwide [61]. Differences in blood culture practice may affect the observed difference in geographical distribution of serotypes. As serotype/group has been shown to be independently associated with IPD severity in adults [32, 34, 35, 38, 63] those countries that perform blood cultures only on severe hospitalised cases would have higher apparent prevalence for certain serotypes than regions that perform blood cultures on both febrile inpatients and outpatients. For example, serogroup 1 has similar, low isolation rates (0.9 per 100,000 person-years) in the US and Europe [44]. However serotype 1 causes only 0.5% of IPD in the US (where blood cultures are performed routinely on febrile inpatients and outpatients) and 5% of reported IPD in western Europe where outpatient blood cultures are performed uncommonly suggesting this serotype is associated with more severe disease [63]. These differences in blood culturing pratice between countries may also account for the differences in serotype 1 incidence between industrialised and developing countries Pneumococcal Pneumonia Pneumococcal pneumonia is estimated to cause one million deaths in children <5 years each year [64, 65]. However, this estimate is based on the contribution of S. pneumoniae to acute respiratory infections (ARI) in studies using children who mostly survived, which does not account for the role of pneumococcus in fatal pneumonia [66]. Determining the aetiology of pneumonia is difficult, and thus studies that calculate an incidence rate for pneumococcal pneumonia are fraught with inaccuracy [67]. There are only a few studies, mostly from developed countries, that have documented pneumococcal pneumonia and are largely recognised as underestimates due to the insensitivity of diagnostic methods used, which are usually blood cultures [65]. The studies vary in terms of sample size, case definitions, methods used to determine aetiology, and the age of subjects included. One of the best studies was from the Gambia where a randomised placebo-controlled trial in the Gambia was undertaken randomising infants to receiving either 3 doses of the 9-valent

43 8 pneumococcal conjugate vaccine or placebo. When using the vaccein as a vaccine probe, the preventable burden of clinical and radiological pneumonia was found to be about 15 episodes per 1,000 child-years [68]. This figure is likely to be an underestimate as many chest X-rays were not acquired in children seen in the first year of the study [68]. Two other studies were from countries that could be classified as developed. Israel is classified by UNICEF as both developed and developing [69], and Chile is considered to be newly industrialising [70]. As a result, the rates in both of these countries were closer to those from developed countries, rather than those from the other developing countries. The rates are likely to be underestimates as blood cultures remain a highly insensitive method of diagnosing pneumococcal pneumonia [71-81]. Even with the most sophisticated laboratory procedures, only a small proportion of pneumonia infections are bacteraemic [74]. Children may present to private general practitioners and receive antibiotics prior to hospital presentation. Poor laboratory facilities, inadequate transportation of specimens to the laboratory, and poorer access to health care affect bacterial isolation rates [70, 82]. It is very difficult to estimate CFR for pneumonia. In countries with good access to health care, CFRs are usually lower because children are readily given antibiotic therapy to which they generally respond rapidly [67]. Where access to health care is poor, many children die at home without receiving medical attention and their deaths often go unrecorded. Vaccine probe studies, which compare mortality caused by pneumonia in groups who do and do not receive pneumococcal vaccination for the purposes of the study, are the most accurate way to demonstrate the proportion of pneumonia deaths associated with pneumococcal infection. There is only one randomised controlled trial of the 9-valent pneumococcal conjugate vaccine from the Gambia assessing the impact of the vaccine on all-cause mortality in children. After the trial was redesigned, the impact on all-cause mortality became a secondary objective of the study and the study had become insufficiently powered to address this. Nevertheless, the study found that vaccinated children had a 16% (95% CI, 3-28%) reduction in all-cause mortality [68] Burden of Pneumococcal Disease in Fiji Our Fiji Pneumococcal Project (FiPP) has studied the burden of pneumococcal disease since This included a cross sectional study of OM in young children, a retrospective study of the burden of chest-x-ray (CXR) confirmed hospitalised pneumonia in children <5 years of

44 9 age, a study of the burden of meningitis in children <5 years, and a study of the outcome of pneumococcal and other bacterial meningitis assessing the quality of life in survivors Burden of OM A cross sectional survey of healthy children aged 3 to 13 months attending maternal and child health centres for immunisations was performed from a selection of 8 urban and 11 rural health centres. Risk factors associated for NP carriage and OM were documented. Immunisation status, birth-weight, and current weight were confirmed by examining the child s health card. Children had otoscopy, video otoscopy, and tympanometry performed by 2 trained staff. Seven hundered and seventy-four children (69% Indigenous Fijian and 25% Indo-Fijian, 6% other) were enrolled in the study (median age 7.7 months, range months). The prevalence of OM in either ear was: acute OM 14%, OM with effusion 26%, and chronic suppurtive OM 0.4%. Independent risk factors of OM included being Indigenous Fijian, malnourished, or having symptoms of an ARI Burden of Pneumonia To calculate the incidence and document the clinical features of CXR confirmed pneumonia in children aged between one month and 5 years living in Greater Suva, Fiji a retrospective review was undertaken of children aged between one month and 5 years with a discharge diagnosis suggesting a lower respiratory tract infection (LRTI) admitted to the only admitting hospital, Colonial War Memorial Hospital (CWMH) in Suva, Fiji in the first 10 days of each month from 1st January 2001 to 31 st December 2002 [83]. Clinical data were collected and CXRs were re-read and classified according to WHO standardised criteria for CXR-confirmed pneumonia [84]. Two hundred and forty-eight children with LRTI met the inclusion criteria. CXRs were obtained for 174 (70%) of these cases, of which 59 (34%) had CXR-confirmed pneumonia. The annual incidence of CXR-confirmed pneumonia was 428 cases per 100,000 children aged between one month and 5 years living in Greater Suva. If a similar proportion of the children for whom CXRs were unavailable were assumed to have CXR-confirmed pneumonia, the incidence was 607 per 100,000. The incidence rate ratio for Melanesian Fijian compared to Indo-Fijian children for all LRTI was 2.5 (95% CI, ) and for CXR-confirmed pneumonia was 29.4 (95% CI, ). This difference in incidence rate ratio may be due to Indo- Fijians seeking health care earlier by private pactitioners and hence negating a hospital admission. The CFR was 2.8% in all children with LRTI, and 6.8% in those with CXR-confirmed pneumonia [83].

45 Annual incidence per 100, A retrospective review of pneumonia admissions (ICD10 J12-18) in all ages was undertaken covering the main island of Viti Levu using the Ministry of Health s (MoH) computerised discharge data for the years 2006 and 2007 (Figure 1). The denominator was taken from the 2007 Census. Data showed a peak age-specific annual incidence of hospitalised pneumonia at both extremes of age (unpublished data). Data from a different retrospective review for the years showed hospitalisation rates for pneumonia to be estimated at 525 per 100,000 for the population overall, and 2,226 per 100,000 in children <5 years of age (J. Passmore, unpublished). Figure 1: The annual incidence of hospitalised pneumonia (ICD10: J12-18) for all ages, for Viti Levu y Age groups (years) Burden of Meningitis A retrospective bacterial meningitis study was undertaken including all children admitted to a single hospital, CWMH, aged from one month to <5 years old with a permanent residential address within a defined catchment area over a 3-year period [85]. Cases of confirmed bacterial meningitis were those with any of: positive bacterial culture from cerebrospinal fluid (CSF); CSF pleocytosis > 100 white cell (WC), or WC with glucose <4.0 mmol/l and protein >100 mg/dl; or latex antigen positive in the CSF and/or Gram stain positive CSF. Individual medical and laboratory records were examined to identify cases. There were 50 cases of culture proven IPD giving an annual incidence of 47.5 per 100,000 <5 years of age. The CFR was 20%. There were 28 cases of pneumococcal meningitis giving an annual incidence of 24.2 per 100,000 <5 years of age. The case fatality rate was 27% [85].

46 11 The quality of life in meningitis survivors was also assessed [86]. There were 37 meningitis cases and 148 healthy controls. Twenty-four percent (n=9) had a history of S.pneumoniae meningitis and 24% had another pathogenic organism. The remaining 52% (n=19) had sterile but purulent CSF. The quality of life score (using a quality of life questionnaire, the PedsQL) was lower in the cases (median 86, IQR 71-97) compared to the controls (median 97, IQR ). The children with a laboratory proven history of pneumococcal meningitis showed a median score of 71 (IQR 55-81). Children with a history of pneumococcal meningitis had a median score more than 10 points lower than children with non-pneumococcal meningitis [86]. 1.6 Protection Against Pneumococcal Disease Host Defense Mechanisms The immune response to S.pneumoniae requires both innate and adaptive components. Once pneumococci have crossed the first natural barrier of the host, they immediately trigger the activation of some components of innate immunity in addition to the deposition of opsonins on the surface of pneumococci. Initiation of the innate response is largely mediated by Toll-like receptors (TLRs) [87, 88]. Early TLR-mediated signaling results in immune cell activation that drives the development of subsequent adaptive immunity, mediated by B and T cells. Complement plays a key role but CRP, lectins, and IgM anticarbohydrate antibodies are responsible for opsonisation. CRP binds phosphorylcholine in the pneumococcal cell wall and activates complement. The killing of S.pneumoniae requires opsonisation by a serotype-specific antibody together with complement, followed by phagocytosis by neutrophils and macrophages. Neutrophil chemotaxis is mediated by the complement activating properties of pneumococcal components such as pneumolysin, C-PS, and cytokines. Extracellular pneumococci induce the release of pro-inflammatory cytokines (interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, IL-12, and interferon-γ (IFN- γ), and anti-inflammatory cytokines IL-4 and IL-10 [89]. Pro-inflammatory cytokines increase rapidly in response to the release of pneumococcal cell wall components during autolysis. Cytokines trigger a complex cascade of inflammatory mediators that regulate the various arms of the inflammatory response. Phagocytic cells as well as NK cells and T-cells play an important role in the elimination of pneumococci from alveoli via the production of chemotactic and regulatory cytokines. Infants elicit weak immunological responses to the encapsulated pneumococcus due to the thymus independent (TI) nature of this bacteria. TI antigens do not require T-cells to induce

47 12 an immune response [90]. TI antigens do not or poorly induce immunologic memory. The antibodies that are produced are primarily of the IgM isotype and are produced in lesser quantities IgG2 [91]. The TI antigens are further divided into 2 categories based on their interaction with B cells: type 1 (TI-1) and type 2 (TI-2) antigens [90-92]. TI-1 antigens induce proliferation and differentiation of B cells in adults but also neonates. In contrast, TI-2 antigens induce a limited immune response in children <2 years of age, but older children and adults repond to TI-2 antigens with the formation of antibodies by activated B cells. Pneumococcal polysaccharide is a TI-2 antigen. The spleen is important in the immune response to pneumococci as it contains both antibody-producing B cells and phagocytes [93]. The splenic marginal zone at the junction of the red and white pulp is an important site of host defense against bacterial infection. The marginal zone contains macrophages, dendritic cells, and B cells and provides the first line of defense against blood-borne pathogens. Polysaccharide antigens preferentially localise in the marginal-zone B cells found only in the spleen. These B cells are present in low numbers at birth [94] and only appear with adult features after 2 years of age. Children <2 years of age have quantitative defects in IgG2 and IgG4 isotypes [95] with IgG2 isotype being considered as the most effective immunoglobulin against some polysaccharides [96]. Humoral responses are currently considered the major adaptive mechanism for bacterial clearance [97] although there is growing evidence of the role of cell-mediated immunity in the protection from NP colonisation [98-100]. It has been well demonstrated that antibodies to the pneumococcal capsule are sufficient to protect against IPD, it is less clear whether they are necessary or they constitute the primary mechanism of natural resistance to pneumococcal infection [101]. In the absence of PCV in a population, IPD incidence naturally declines approximately 2 years prior to the age at which unimmunised children show a rise in serum anti-capsular antibody. In the pre-pcv era anti-capsular antibody was shown not to be above the putative protective level of 0.35µg/mL by 36 months of age in US children, yet disease from the common serotypes, 6 and 14, are almost 10 fold lower than at 12 months of age [ ]. This indicates that there are other protective mechanisms apart from serum anti-capsular antibody that confer protection. NP carriage can lead to serotypespecific acquired immunity to pneumococcal carriage for some serotypes [104]. However a recent longitudinal study in Bangladeshi infants showed a reduced rate of NP pneumococcal acquisition in a manner consistent with the induction of serotype-independent protective immunity [105]. Protection may be derived from the development of CD4+ T cells of the IL-

48 13 17A lineage that recognise pneumococcal antigens that are expressed during colonisation [106, 107]. Secretion of IL-17A from these cells may recruit phagocytes to the colonisation site and help reduce the duration of carriage [101]. The critical role of pneumococcal-specific memory B cells in the first line of defense against pneumococcal infection has recently become an important area of research. IgM + CD27 + and switched IgG + CD27 + memory B cells are involved in the immune response to the 23-valent pneumococcal polysaccharide vaccine (23vPPS) since these cell populations are deficient in patients with primary immunodeficiency syndromes who are susceptible to recurrent infections with encapsulated bacteria [108, 109]. Infant B cells are unable to recruit cognate CD4 + T-cell help through T-cell receptor recognition of peptide-major histocompatibility complex class II complexes on the surface of antigen presenting cells [110]. CD4 + cells contribute to the early host resistance to infection as shown by an early rapid T-cell infiltration to areas that are subject to increased pneumococcal invasion. Pneumolysin seems to be responsible for the pattern of T-cell infiltration [111]. Data suggests that pneumolysin induces the production of IFN- γ and TNF-α in peripheral blood mononuclear cells and IFN- γ and IL-10 in adenoidal mononuclear cells [112]. CD4 + T-cell proliferative responses to pneumolysin are significantly higher in children who do not have detectable pneumococcal NP carriage which suggests that natural CD4 + T-cell immunity to pneumococcal protein antigens could modulate NP carriage [112]. However it is unclear whether this T-cell immunity cleared existing carriage or prevented new colonisation Pneumococcal Vaccines In 1911, Wright et al developed a crude whole-cell pneumococcal vaccine to immunise South African miners [113], a group with a very high pneumococcal burden and mortality. A number of other clinical trials were conducted in South Africa by Spencer Lister and others abroad on the safety and efficacy of polysaccharide vaccines [ ]. However the validity of the results were questioned due to various methodological flaws in study design such as the lack of randomisation and inadequate clinical follow up. Polyvalent vaccine trials were continued in the 1940s and 2 hexavalent vaccines were commercially produced and licensed in the late 1940s. However effective antibiotics, sulphonamides from 1937 and penicillin in the mid 1940s, were soon available and with subsequent improvements in patient outcomes the interest in pneumococcal vaccine development waned. Data from the US in 1964 told a different story and it was estimated that despite effective antibiotic availability the CFR of pneumococcal bacteraemia was up to 25% [117]. This led to the relatively rapid

49 14 development of a polyvalent pneumococcal vaccine in the 1970s which was evaluated in South African gold miners and found a protective efficacy of 76-92% [118, 119]. These findings led to the licensure of the 14-valent pneumococcal polysaccharide vaccine (PPS) in the US in 1977 which was replaced by the 23vPPS in PCV was the first pneumococcal conjugate vaccine to be licensed. Vaccine manufacturers are developing different formulations containing more than 7 capsular polysaccharides and the newly developed 10- valent pneumococcal conjugate vaccine (with the addition of serotypes 1, 5 and 7F) and the 13-valent pneumococcal conjugate vaccine (with the addition of serotypes 1, 3, 5, 6A, 7F, and 19A) have recently been licensed in a number of countries in 2009/2010. In addition, Merck Inc. has a 15-valent pneumococcal conjugate vaccine under advanced stages of development Measurement of Immune Responses to Pneumococcal Vaccines Given the difficulties related to performing new vaccine efficacy trials where a product is already licensed and in widespread use, WHO has developed a number of serological criteria based on demonstrating non-inferiority compared with the licensed PCV, to assist regulatory agencies in evaluating new pneumococcal conjugate vaccine formulations. The primary criteria for the evaluation of new pneumococcal conjugate vaccines are [102]: 1. The primary endpoint should be the serotype-specific IgG antibody concentration as measured by enzyme-linked immunosorbent assay (ELISA), using 22F preabsorption, 4 weeks following a 3 dose primary series for all pneumococcal serotypes; and 2. The proportion of vaccinees with antibody concentrations 0.35μg/mL for all pneumococcal serotypes; The secondary supportive criteria are the demonstration in at least a subset of vaccinees of: 3. Functional activity: Opsonophagocytic activity (OPA) as measure by opsonophagocytic assay following a 3 dose primary series compared ideally to unvaccinated age-matched controls; and 4. Immunologic memory: Evidence of memory shown by administration of a booster dose of 23vPPS compared with unvaccinated controls; and/or antibody avidity. A recent review has recommended that conjugate vaccine as the booster be used instead of 23vPPS due to the issue of hyporesponsiveness [120].

50 Measurement of pneumococcal antibody concentrations As clinical protection against pneumococcal infection is mediated by antibodies to the capsular polysaccharide, an ELISA that measures serotype-specific IgG antibody concentrations is the accepted measure of immunogenicity. The third-generation ELISA assay was adopted in a meeting convened by WHO in 2000 [121] following the finding that the second-generation ELISA using the single absorption method with C-PS, had insufficient specificity [122]. Some sera contain polyreactive antibodies that recognise pneumococcal polysaccharides of different serotypes resulting in cross reactivity [123, 124]. The ELISA specificity has been improved by pre-absorption not only with C-PS but also with pneumococcal 22F capsular polysaccharide [123, 125]. In addition, this additional step has increased the correlation between anti-capsular polysaccharide antibody concentration and OPA [123, 126]. GlaxoSmithKline believe overnight incubation with 22F as opposed to 30 minute incubation as per the WHO method produces more accurate data at lower levels and 0.2μg/mL is equivalent to 0.35μg/mL [127]. The anti-pneumococcal antibody concentrations required for protection for an individual are not known. A concentration of IgG anti-capsular polysaccharide antibodies 0.35μg/mL (without using the 22F absorption step) measured 4 weeks following a primary series is recommended as the protective level at the population level [102]. This pooled estimated is based on the IPD efficacy data from 3 double-blind randomised controlled trials undertaken in Northern California [103], American Indians [128], and South African infants [129]. If a high proportion of the vaccinated population achieves antibody concentrations 0.35μg/mL then a high level of protection from IPD can be predicted in that population [130]. A reanalysis using the double absorption method (with C-PS and 22F reabsorption) found the protective concentration to be marginally lower at 0.32μg/mL [130] Functional activity The primary mechanism for protection against IPD is mediated by the presence of opsonophagocytic antibodies [131]. Antibodies to pneumococcal polysaccharide offer protection by acting as an opsonic factor and activating complement thereby promoting phagocytosis of pneumococci. The ability of antipneumococcal antibodies to induce phagocytosis and killing is believed to be a surrogate for indicating protective activity. As both antibody concentration and avidity contribute to OPA and confer protection in a mouse challenge [ ], a positive OPA is considered to indicate good vaccine derived protection. While it is recommended that antibody concentration, as measured by a standardised third generation ELISA, be the primary

51 16 measure when licensing new pneumococcal conjugate vaccines [102], OPA has been accepted as a necessary additional measure and the reference method for measuring the protective capacity of pneumococcal antibodies [126]. The killing OPA method, which measures the killing of live bacteria is regarded as the most biologically relevant method [126, 136]. There are a number of different methods available and there has been extensive efforts to standardise the OPA assay [120]. OPA assays are labour-intensive and difficult to perform with large numbers of samples. A number of multiplexed assays have been developed using fluorescent dye to quantify killing [137], using bacteria or latex beads coated with different polysaccharides [138], or the use of pneumococcal strains resistant to clinically irrelevant antibiotics to quantify killing [137, 139, 140]. In general, the correlation between antibody concentration and OPA seems to be good in infants who have been immunised with conjugate vaccines [141, 142]. Correlates have been high for vaccine serotypes (VT), but lower for cross-reacting serotypes like 6A and particularly 19A [143] Immunologic memory Antibody concentrations alone failed to accurately predict the success of Haemophilus influenzae type b conjugate vaccines (Hib) [144] as conjugate vaccines not only induce antibodies but also prime the immune system for later protective memory responses. As this is also true with pneumococcal conjugate vaccines, memory may also play an important role in protection from pneumococcal disease. Immunologic memory to a polysaccharide antigen can be defined as a response that is present in an otherwise non-responsive individual such as infants, characterised by a higher antibody response that is dominated by IgG on exposure to an antigen, and characterised by antibodies with increased avidity as a result of affinity maturation [145]. Antibody affinity describes the strength with which an antibody binds to a complex antigen. The antigen binding capacity of polyclonal antibodies with different affinities can be measured as antibody avidity. Antibody avidity is an expression of the functional antibody affinity and may affect the protective efficacy of antibodies. In vitro, higher avidity antibody is associated with greater opsonophagocytic capacity [ ]. Furthermore, findings from a study assessing the contribution of avidity, antibody concentration, and IgG subclass to opsonophagocytic activity demonstrated that lower amounts of high avidity antibody were sufficient for killing of bacteria whereas higher amounts of low avidity antibody were required for effective killing activity [147]. While the importance of avidity in determining protection from disease is unclear, studies of Haemophilus influenzae type b (Hib) conjugate

52 17 vaccine have demonstrated that antibody avidity is strongly associated with functional activity of anti-hib polysaccharide antibodies [149, 150], and anti-hib polysaccharide antibodies of high avidity have been found to have protective efficacy in experimental Hib infection [151]. Immunologic memory needs to be demonstrated for the licensure of new pneumococcal conjugate vaccine formulations by either the administration of a booster dose of 23vPPS and the measurement of the increase in IgG concentration, and/or assessment of antibody avidity [102]. The measurement of antibody avidity following PCV immunisation provides information on both the development of B cell memory [152] and the functional activity of antibodies [132, 153]. One method of demonstrating immunological priming is to assess the ability of 23vPPS to boost the immune response and elicit antibodies with increased affinity. Differences in affinity maturation have been used to demonstrate differences in priming capacity of some pneumococcal conjugate vaccines [147, ] and in reduced dose schedules [157]. Although qualitative changes in antibody response have been demonstrated following conjugate vaccine administration, the clinical relevance of these changes is unclear. In ELISA techniques, the binding of antibody to the coated antigen may be prevented by competitive inhibition using decreasing concentrations of free antigen by a dissociating agent such as thiocyanate. Thiocyanate interferes with the antibody-antigen binding. The elution assays are based on the dissociation of antigen-antibody complexes of low avidity. Avidity assays have not been standardised and their value in predicting protection remains to be determined Pneumococcal conjugate vaccines Infant B cells are immunologically immature, and respond poorly to TI polysaccharide antigens. However, in the presence of activated T cells, they can be stimulated to produce both antibody-producing plasma cells and memory cells [158]. By covalently conjugating a protein carrier to bacterial capsular polysaccharide, this antigen is able to induce a T cell-dependent antibody response. This proved effective in developing vaccines for Hib, and has now proven safe and effective for pneumococcal conjugate vaccines. Currently, PCV is marketed internationally and is included in over 70 countries national immunisation programmes and on the private market in many more. WHO considers PCV as

53 18 a priority in national immunisation programmes, particularly in countries where mortality is high (>50/1,000 live births) or where >50,000 children die annually [4]. Seven serotypes (1, 5, 6A, 6B, 14, 19F, 23F) account for approximately 58-66% of all IPD worldwide [61]. The PCV coverage rate of IPD serotypes in young children in industrialised countries tends to be higher (65-80%) [4]. The US, New Zealand, and Australia reported similar coverage by PCV (80%, 81%, and 84% respectively) for IPD isolates in young children pre-vaccine introduction [45, 51, 54]. Rural Thailand and Nigeria have similar but lower PCV coverage rates for IPD (51% and 55% respectively) [50, 159]. In general, the range of serotypes causing disease in affluent countries like the United States and in Europe is relatively narrow and largely confined to the serotypes found in PCV (4, 6B, 9V, 14, 18C, 19F, 23F). A recent review found that if a pneumococcal conjugate vaccine with as few as 6-7 serotypes (including serotypes 1, 5, and 14, and assuming that 6B provides cross protections against 6A disease) were developed it would cover at least 60% of all IPD worldwide. Furthermore, this review stipulated that vaccines should include serotypes that account for at least 60% of the IPD isolates among children in the region for which they are proposed [61]. Each 0.5mL dose of PCV contains 2 μg of polysaccharide for serotypes 4, 9V, 14, 19F, and 23F; 2 μg of oligosaccharide 18C, and 4 μg of polysaccharide type 6B. These saccharides are individually conjugated to diphtheria CRM 197 protein. CRM 197 is a nontoxic variant of diphtheria toxin. WHO recommends 3 intramuscular doses given at 6, 10, and 14 weeks of age at least 4 weeks apart. A booster dose administered after 12 months of age will improve the immune response [4]. Some European countries including the UK have introduced a 2+1 schedule with 2 doses given during infancy with the third dose given towards the end of the first year of life Safety PCV is safe and well tolerated [4]. More than 198 million doses have been distributed worldwide [160]. The PCV safety profile was evaluated in clinical trials prior to licensure in more than 18,000 infants in the US [103]. Local injection site reactions are not uncommon but are generally mild and self-limiting [103, ]. Two years following licensure and national introduction into the US, post-marketing surveillance further evaluated the safety profile of PCV [166]. The majority of reports in the first 2 years after licensure of PCV described generally minor adverse events previously identified in clinical trials [166]. A retrospective cohort study of adverse events requiring medical attention found a slight increase in reactive airways disease among infants vaccinated with PCV

54 19 compared with a historical control of infants vaccinated with Hib vaccine [167]. A similar study design found children with Kawasaki disease having a 2 fold increased probability of having received PCV which was not statistically significant when adjusted for sex, age, race, and other factors [168]. Further evaluation showed no association between Kawasaki disease and PCV [169] Immunogenicity The standard PCV immunisation schedule that has been studied has been a 3 dose primary series with or without a booster in the second year of life. The geometric mean concentrations (GMC) one month following a 3 dose primary series in infancy from the trials using PCV or the 5 or 9-valent pneumococcal conjugate vaccine (with the same carrier protein as PCV and the additions of serotypes 1 and 5) are shown in Table 1. Direct comparisons between studies are difficult as the vaccination schedules and the laboratories and techniques were different in different settings. In general, a low antibody response was elicited after the first dose, substantial antibody responses were seen after subsequent doses and a clear booster type response to a dose in the second year of life. For all serotypes good post-primary series antibody responses were elicited and serotypes 6B, 9V, and 23F tend to be less immunogenic overall [163, ]. However for serotypes 6B and 23F following a booster in the second year of life the antibody concentrations were high. Antibody responses in Finnish infants were approximately 2-3 times higher than infants in the US. In South African infants, antibody concentrations against serotype 6B were approximately 4 times higher than either Finnish or US children. Differences in serotypespecific responses have been found in other populations, and this has been postulated to be due to the priming effect of tetanus toxoid given to women in pregnancy in developing countries (when tetanus-conjugated polysaccharide vaccines were administered to infants), a nonspecific BCG vaccine effect, early pneumococcal NP acquisition, and genetic differences amongst the populations [174]. An important characteristic of the pneumococcal conjugate vaccines are their ability to create immunologic memory. Antibody concentrations achieved after primary vaccination usually decline during subsequent months. However booster immunisation with either 23vPPS or conjugate induces a marked increase in antibody concentrations in children who have been primed with conjugate vaccine in infancy. Another feature of the anamnestic response produced by conjugate vaccines is the rapidity of the antibody response within 7 to 10 days after the re-vaccination [173, ]. IgG antibodies tend to dominate the response. These findings indicate that conjugate vaccines prime the infant immune system

55 20 to elicit a robust response on subsequent exposure to either polysaccharide or conjugate vaccine. There have been several studies involving children in a number of countries using different pneumococcal conjugate formulations and schedules, comparing the immunogenicity of a 23vPPS or PCV booster following a pneumococcal conjugate vaccine primary series. The majority of studies have shown that serotype-specific antibody concentrations are generally higher following 23vPPS than PCV booster [157, 172, ]. The higher response may be due to the higher dose of pneumococcal polysaccharide in the 23vPPS, compared to PCV, enhancing the stimulation of memory B cells or by stimulating a greater number of B cells overall [189]. Despite higher antibodies generated post PCV/23vPPS a study comparing PCV/23vPPS with a PCV/PCV schedule found similar vaccine efficacy results against acute OM [188]. Studies have found that a conjugate vaccine booster is better at increasing antibody avidity than a PPS booster [147, 154, 157, 190]. Infants primed with PCV and who received a PCV booster, but not PPS booster, had an increase in avidity of anti-pneumococcal polysaccharide [154] which suggested the response to PCV was T cell dependent, but the T cell-independent PPS only triggered existing memory B cells. A study in infants immunized at 2, 4, and 6 months of age with one of 4 different conjugate vaccines, and boosted at 14 months with the homologous conjugate or PPS found that the avidity of serotypes 6B, 14, 19F and 23F increased with age, but only a booster dose of conjugate further increased avidity compared to a PPS booster [147]. Similarly, a reduced dose study in the UK found that a booster with PCV gave significantly higher avidity for the 3 serotypes tested (6B, 14, 23F) compared to a 23vPPS booster [157]. Avidity for 3 serotypes tested in Ghanaian children was higher in those who had received PCV rather than 23vPPS [190]. This phenomenon may be due to the fact that conjugate boosters promote affinity maturation by T cell dependent mechanisms. An alternative explanation for the differences in avidity is that the amount of antigen in the PPS is high enough to induce both high and low avidity B cell clones to produce antibodies, whereas only high avidity clones are induced by lower concentrations of polysaccharides in conjugate vaccines [191].

56 21 Table 1: Summary of serotype-specific GMC data from trials of CRM197-conjugated pneumococcal vaccines, one month post primary series Ref. Country PCV Schedule Co-administered parenteral vaccines n GMC (μg/ml) B 9V 14 18C 19F 23F [163] Canada 7v 2,4,6m DTaP-IPV-Hib, HBV [192] Finland 7v 2,4,6m DTwP-Hib [193] France 7v 2,3,4m DTwP-IPV/Hib [162] Germany 7v 2,3,4m DTaP-HBV-IPV/Hib [165] Germany 7v 2,3,4m DTaP-HBV-IPV/Hib [164] Germany 7v 2,3,4m DTaP-IPV-Hib (GSK) [194] Iceland 9v- MCC 3,4,5m DTaP-IPV/Hib [195] UK 9v-MCC 2,3,4m DTPwP/Hib [157] UK 9v 2,3,4m DTaP-Hib, MCC [172] UK 7v 2,3,4m DTwP, Hib [196] US 7v 2,4,6m DTaP-HBV-IPV, Hib [103] US 7v 2,4,6m DTaP

57 22 Ref. Country PCV Schedule Co-administered parenteral vaccines n GMC (μg/ml) B 9V 14 18C 19F 23F [173] US 7v 2,4,6m DTwP-Hib, HBV [182] US 7v 2,4,6m DTwP/Hib [197] US Navajo/White Mountain Apache 7v 6w,4,6m DTaP, Hib, IPV, HBV [177] Bangladesh 7v 18,24,28w DTwP-Hib [198] South Africa 9v 6,10,14w DTwP-Hib, HBV [199] South Africa (HIV-) v 6,10,14w DTwP, Hib, HBV [200] The Gambia 9v 2,3,4m DTwP-Hib, HBV [179] The Gambia 9v 2,3,4m DTwP, HBV [201] The Gambia 5v 2,3,4m DTwP, HBV [202] The Gambia 9v 6,10,14w DTwP-Hib, HBV IPV and Hib co-administered with 1 st and 2 nd dose of PCV only; HBV co-administered with 1 st dose of PCV only 2 Details of other co-administered vaccines and vaccine combinations unknown 3 DTwP-Hib and OPV co-administered with 1 st dose of PCV only 4 HBV co-administered with 1 st and 3 rd dose of PCV only MCC Meningococcal conjugate vaccine

58 Efficacy In a large randomised controlled trial in the US, PCV given at 2, 4, and 6 months of age, with a fourth dose in the second year of life, was immunogenic and provided 97.4% (95% CI, %) protection against VT IPD in infants who received at least 3 doses of the vaccine [103]. In the intent-to treat analysis, for children who had received at least one dose of the vaccine there was an 89.1% (95% CI, %) reduction against VT IPD overall [103]. In the Gambia, a randomised controlled trial of 3 doses of the 9-valent pneumococcal conjugate vaccine (with the addition of serotypes 1 and 5) in infancy had an efficacy of 77% (95% CI, 51-90%) against VT IPD and 45% (95% CI, 19-62%) efficacy against all IPD serotypes [68]. In South Africa, the 9-valent pneumococcal conjugate vaccine given at 6, 10, and 14 weeks of age and had 83% (95% CI, 39-97%) protective efficacy against VT IPD in HIV-negative children and 65% (95% CI, 24-86%) efficacy in HIV-positive children [129]. There was 35% (95% CI, %) vaccine efficacy against all IPD serotypes [129]. In a group-randomised study in Navajo and White Mountain Apache Indian children younger than 2 years, the per protocol primary efficacy of PCV against VT IPD was 76.8% (95% CI, %) and the intention-to-treat efficacy was 82.6% (95% CI, %), after group randomisation had been controlled for [128]. As most cases of pneumococcal pneumonia are non-bacteraemic, the lack of a sensitive and specific endpoint to determine vaccine efficacy for pneumococcal pneumonia has resulted in the use of radiologically defined pneumonia as an endpoint in clinical trials [84]. However this endpoint may under-estimate the true public health benefit of pneumococcal conjugate vaccines [203, 204]. Using the WHO method, the efficacy against first episode of radiographically confirmed pneumonia adjusting for age, gender, and year of vaccination for children from the US who received 3 doses of PCV in infancy followed by a fourth dose at months of age, was 25.5% (95% CI, %) for intent-to-treat and 30.3% (95% CI, %) for the per protocol analysis [205]. The 9-valent pneumococcal vaccine trials in the Gambia and South Africa described previously, documented a vaccine efficacy against the first episode of radiologically confirmed pneumonia in the per protocol analysis of 37% (95%CI, 27-45%) and 25% (95%CI, 4-41%) respectively [68, 129]. In the South African and Gambian trial, the efficacy estimate against all clinically diagnosed pneumonia was similar at 7% (95% CI, -1-14) and 7% (95% CI, 1-12) respectively [68, 129]. In addition, vaccinated Gambian children showed a 16% (95% CI, 3-28%) reduction in all-cause mortality [68]. In the Philippines, children who had received 3 doses of an 11-valent pneumococcal conjugate vaccine (with the addition of serotypes 1, 3, 5, and 7F) showed a vaccine efficacy of 22.9%

59 24 (95% CI, ) against radiologically confirmed pneumonia in <24 month olds and a 34% (95% CI, %) reduction in <12 month olds [206]. There was no efficacy demonstrated against clinical pneumonia defined as cough and fast breathing [206]. Comparing OM outcome between studies is more challenging as the definitions of a case of OM varies between studies. However a Cochrane review pooling results from 4 trials in disparate subjects ranging from healthy infants or toddlers, to toddlers with recurrent OM, found a small effect (RR 0.921; 95% CI, ) on the prevention of acute OM [207]. A study in Finnish infants found that PCV had a small and non-significant effect (6% reduction; 95% CI, -4-16%) on all OM [161]. These children were followed up at 4 to 5 years of age and the vaccinees were found to have a 39% (95% CI, 4-61%) reduction in tympanostomy tube placement [208]. In US children, the PCV vaccine efficacy against any OM episode using per protocol analysis was 7% (95% CI, %) and for tympanostomy tube placement was 20.1% (95% CI, %) [103]. Children from this study were followed for up to 3.5 years and PCV was found to reduce OM visits by 7.8% (95%CI, %) and tympanostomy tube placements were reduced by 24% (95% CI, 12-35%) [209]. The vaccine efficacy of a 7-valent pneumococcal conjugate vaccine conjugated to meningococcal outer membrane protein complex was similar to PCV, and a booster with 23vPPS had similar efficacy to the conjugate booster despite higher antibodies generated following the PCV/23vPPS combination [188]. The vaccine efficacy against acute OM in infants who had received an 11-valent pneumococcal conjugate vaccine with each serotype conjugated to Haemophilus influenzaederived protein D at 3, 4, 5, and months was 33.6% (95% CI, %) [210]. This increased benefit over the other trialed conjugate vaccines may be due to the H.influenzaederived protein D carrier protein which provided efficacy against non-typable H.influenzae, a common cause of acute OM but this remains to be proved [210] Effectiveness Since the introduction of PCV into the national immunisation schedule in the US in 2000, there had been an impressive 69% reduction in IPD in children <2 year old [52]. Similarly in Australia, Germany, Portugal, Spain, Denmark, and Norway significant declines in IPD rates in young children have been reported [ ]. The IPD rate in the high risk White Mountain Apache children has declined to its lowest rate ever since the introduction of PCV [217]. An observational study in the US using routine administrative data found that following PCV introduction the average annualised pneumococcal meningitis rate in children <2 years old decreased by 66%, and had declined in children 2 to 4 years old and those 65 years old compared with rates pre vaccination [218]. For those children in

60 25 the US who developed IPD despite being vaccinated, IPD resulted primarily from NVT, or in children who were incompletely vaccinated or had co-morbid conditions [219]. Approximately 4 years following PCV introduction in the US replacement IPD, particularly due to serotype 19A, developed due to capsular switching and clonal expansion [220, 221]. Serotype 19A has become the predominant cause of IPD in children [ ] and serogroups 15 and 33 have been reported as increasing causes of IPD in children in a multicentre study in the US [225]. The annual incidence of NVT IPD has significantly increased in the <5 year olds and 65 year olds in one study [222] and in children and adults in another [226]. Serotype replacement IPD is more common in the immunocompromised population but occurs in both the vaccinated group and older age groups [227]. Despite there being a significant reduction overall in pneumococcal meningitis in the US, a recent study has shown that the rate of NVT meningitis has significantly increased from 0.32 to 0.51, an increase of 60.5% (p<0.001) and the proportion of penicillin non-susceptible pneumococcal meningitis isolates has increased significantly compared with pre-vaccine levels [228]. The success of the introduction of PCV into the UK s national schedule has been comprised to some extent by rapid NVT replacement [229]. The impact of PCV introduction on hospitalised pneumonia was investigated in an interrupted times series analysis that used all-cause pneumonia and pneumococcal pneumonia admission rates as the main outcome [230]. The advantage of this approach was that it could account for seasonal and secular trends that were present before the intervention [231]. Admission rates were compared before and after the introduction of PCV and rates of dehydration were used as a comparison. All-cause pneumonia rates had declined by 39% (95% CI, 22-52%) and pneumococcal pneumonia rates declined by 64% (95% CI, 47-77%) [230]. Dehydration rates remained steady throughout the study period. However biases including differences in physician diagnoses, coding, or admission practice following the introduction of PCV may have contributed to these findings. A study in the US assessing the impact of PCV on inpatient pneumonia rates found a suggestion of a decrease (IRR 0.6, p=0.07) in infants in the post- introduction period [232]. This same study found a significant decrease in the risk (IRR, 0.74, p=0.02) of confirmed outpatient pneumonia in the period after PCV introduction compared to the period prior to PCV introduction [232]. In contrast, another study found PCV to have no impact on the rate of outpatient pneumonia presentations in the US in children <2 years of age [233]. In Australia, a recent study using a national electronic database found that after adjustment for background and seasonal

61 26 trends, a reduction of 38% (95%CI, 36-40%) and 29% (95%CI, 26-31%) in all-cause pneumonia was found following PCV introduction (3 infant PCV doses with no booster) in children <2 years and 2 to 4 years respectively [234]. In contrast, recent studies have found an increase in empyema hospitalisations in US children [235, 236]. Using nationally representative dataset in the US, one observational study found that empyema hospitalisations of children 18 years old increased almost 70% and complicated pneumonia (including empyema, pleural effusion, or bacterial pneumonia requiring a chest tube or decortication) increased compared to the pre-vaccine era despite the rate of all hospitalised bacterial pneumonia and IPD significantly decreasing [235]. These findings, if real, may reflect increases in serotype replacement causing NVT pneumococcal pneumonia, reflecting that NVT may be more pathogenic than VTs, or an increase in complicated pneumonia due to other bacterial pathogens occupying pneumocci s former niche and causing disease. Similarly an ecologic study using a different national dataset in the US found a doubling of pneumonia admissions complicated by empyema in the postvaccine era with an increase in streptococcal and staphylococcal empyemas [236]. However this upward trend in empyema admissions was found prior to PCV introduction [236]. National outpatient OM visit rates were compared before and after the introduction of PCV in the US [233]. OM visit rates significantly declined by 20% (p=0.014) in children <2 years of age [233]. The risks of developing frequent OM or having tympanostomy tubes inserted were compared in children <2 years of age from 2 states in the US before and after the introduction of PCV [237]. Frequent OM visits declined by 17% and 28%, and tympanostomy tube insertion declined by 16% and 23% in Tennessee and New York children respectively [237]. In Australia there was a significant reduction in tympanostomy tube insertion in children up to 2 years of age following the introduction of PCV in the national immunisation schedule [238]. In a before and after PCV introduction study in a very high risk population of Australian Indigenous infants, it appeared that no benefit was found of PCV on OM rates althoughthe authors state bias and confounding may have impacted on the findings due to the nature of the design of the study [239]. A small prospective study in the US documenting the otopathogens in children 6 to 36 months of age with receurrent OM and children with infrequent acute OM found that 6 to 8 years post PCV introduction, NVT had replaced VT, and the frequency of isolation of S. pneumoniae was nearly equal to that of non-typeable Haemophilus influenzae, with other pathogens being less frequently isolated from middle ear fluid [240].

62 Reduced dose PCV schedules Although 3 PCV doses were originally considered to be required for an optimal immune response to PCV some studies have suggested that a single dose may be sufficient at least where stimulation with high pneumococcal carriage and earlier carrier priming has occurred [198, 241]. Because conjugate vaccines induce priming, it is possible that they will protect even in the absence of a circulating antibody response. This raises the possibility that fewer doses of conjugate vaccine than are presently recommended, perhaps even a single dose, may be sufficient to protect from serious invasive disease or death. When the introduction of PCV into the US national immunisation schedule was met with a global shortage of vaccine, many children received fewer than the recommended 4 doses of vaccine. A case control study documenting the impact of this on IPD due to vaccine serotypes found that one and 2 dose schedules given to infants <7 months of age had an effectiveness of 73% (95%CI, 43-87%) and 96% (95%CI, 88-99%) respectively [242]. Models however have predicted that a single PCV dose given between 5-10 months of age could prevent a significant amount of VT IPD [243]. A South African trial showed a significant and potentially protective antibody response to most serotypes following a single dose at 6 weeks of age with at least 70% of infants producing antibody concentrations >0.15μg/mL after a single PCV dose and at least 95% doing so after 2 doses [198]. A study in Filipino infants with an 11-valent pneumococcal vaccine conjugated to either tetanus protein or diphtheria-toxoid showed that a single dose at 18 weeks of age elicited similar antibody concentrations at 9 months of age compared with those that had received 3 doses [241]. A model estimating the herd effect on IPD incidence predicted that even in situations where PCV coverage is less than 3 or 4 doses, PCV may still induce herd effects [244]. The immunogenicity of 3 versus a 2 dose pneumococcal primary series with different coadministered vaccines, is different in different settings [157, 170, 182, 194, 245, 246] (Table 2). Following 3 doses of the 9-valent pneumococcal conjugate vaccine in Icelandic infants, 7 out of the 9 serotypes had significantly higher post primary antibody concentrations compared to the 2 dose group [194]. However the proportion of infants in the 2 dose group with antibody levels >0.35μg/mL (the estimated protective level) was only significantly lower compared to the 3 dose group for serotype 6B [194]. A randomised controlled trial from Israel in which infants received either PCV at 2, 4, 6, and 12 months of age, 4, 6, and 12 months of age, or at 2, 4, and 6 months of age found that the 2+1 group had lower post

63 28 primary IgG concentrations for serotypes 6B, 14, 18C, and 23F compared to the 3 PCV dose groups [247]. A cohort study from the Netherlands compared a 2+1 PCV schedule (given at 2, 4 and 11 months of age) with a 3+1 (given at 2, 3, 4 and 11 months of age) schedule found that pre-booster levels were comparable for 6 of 7 PCV serotypes (except serotypes 6B) and post-booster levels were comparable for 5 of 7 PCV serotypes (except 6B and 19F) [248]. A study using an 11-valent pneumococcal conjugate vaccine in Israeli infants showed a 2 dose schedule was less immunogenic than a 3 dose post primary series for serotypes 6B, 14, 18C, and 23F with a significantly lower proportion of infants with antibody levels 0.35μg/mL for serotypes 6B, 18C, and 23F. This study used an unlicensed 11-valent pneumococcal conjugate vaccine conjugated to diphtheria and tetanus carrier proteins [246]. PCV given to US infants showed 3 doses were needed to achieve an immunological response for serotype 6B but 2 doses were sufficient for the other 6 PCV serotypes [182]. PCV was less immunogenic for serotype 6B in Italian infants after 2 PCV doses than described in the US and Finland, but similar for the other PCV serotypes [161, 173, 245]. A randomised controlled trial in Denmark, Norway, Slovakia, and Sweden comparing a valent pneumococcal non-typeable H. influenzae protein D-conjugate vaccine schedule (given at 3, 5 and months of age) with a 3+1 schedule (given at 3, 4, 5 and months of age) found for most vaccine serotypes a trend towards lower post-primary and post-booster immune responses in children primed with the 2 dose primary series [249]. In contrast, other studies have found minimal immunological differences between a 3 or 2 PCV dose primary series [157, 245]. In a non-randomised study in UK infants no significant differences in GMC following a 3 or 2 dose schedule were found [157]. There were no significant differences in the proportion of infants achieving antibody concentrations >0.35μg/mL except for serotype 14 for which there was a higher proportions achieved in the 3 dose group [157]. Two doses may well be sufficient to protect against most PCV serotypes. In some European countries and Italy, routine immunisations are given in a 2 dose primary series with a booster at or before the end of the first year of life. Norway introduced a 2+1 PCV schedule (given at 3, 5 and 12 months of age) in 2006 [215]. Following this, the incidence of IPD in children less than 2 years of age rapidly declined and the incidence rate of NVT IPD remained stable [215]. The vaccine efficacy was estimated to be 74% (95% CI, 57-85%) [215]. Liguria, in Italy, found a statistically significant reduction in hospitalisation rates for all-cause (15.2%; 95%CI ) and pneumococcal pneumonia (70.5%; 95%CI, %), and for acute OM (36.4%; 95%CI, %) in children born after the introduction of universal PCV [250]. Effectiveness data from the UK using the 2+1 schedule has shown a

64 29 reduction in all infant IPD [251]. There has been a marked reduction in VT disease [251]. However the incidence of NVT IPD has been increasing and remains a concern [251]. Three doses may be required to protect against serotype 6B as breakthrough cases of 6B have occurred [252]. Ongoing surveillance will determine whether these breakthrough cases will be a significant and consistent finding.

65 30 9v-MCC 3,4,5m DTaP-IPV/Hib [247] Israel 7v 4,6m DTaP-IPV-PRPT v 2,4,6m 1 DTaP-IPV-PRPT v 2,4,6m 1 DTaP-IPV-PRPT [245] Italy 7v 3,5m Table 2: Summary of serotype-specific GMC data from trials of reduced dose pneumococcal conjugate vaccines Coadministered B 9V 14 18C 19F 23F GMC (μg/ml) Ref. Country PCV Schedule n Reduced dose GMC data compared with 3 doses (if performed), one month post primary series [194] Iceland 9v-MCC 3,5m DTaP-IPV/Hib DTaP-HBV- IPV/Hib [170] Sweden 7v 3,5m DTaP-IPV/Hib [157] UK 9v 2,4m DTaP-Hib, MCC v 2,3,4m DTaP-Hib, MCC [253] Mexico 13v 2,4m DTaP-IPV-Hib HBV, rotavirus [201] The Gambia 5v 2,4m DTwP,HBV v 2,3,4m DTwP,HBV

66 31 [198] South Africa 9v 6,(10,14w) 3 DTwP-Hib,HBV 9v 6,10,(14w) 4 DTwP-Hib,HBV 9v 6,10,14w DTwP-Hib,HBV Coadministered B 9V 14 18C 19F 23F GMC (μg/ml) Ref. Country PCV Schedule n Reduced dose GMC data compared with 3 doses (if performed), one month post primary series GMC following primary series dose one or 2 compared with post-dose 3 levels [177] Bangladesh 7v 18,(24,28w) 5 DTwP-Hib v 18,24,28w DTwP-Hib [182] US 7v 2,4,(6m) 7 DTwP/Hib v 2,4,6m DTwP/Hib [197] US Navajo/ White Mountain Apache 7v 6w,(4,6m) 8 DTaP, Hib, IPV, HBV v 6w,4,(6m) 10 DTaP, Hib, IPV, HBV

67 32 Coadministered B 9V 14 18C 19F 23F GMC (μg/ml) Ref. Country PCV Schedule n Reduced dose GMC data compared with 3 doses (if performed), one month post primary series 7v 6w,4,6m DTaP, Hib, IPV, HBV GMC from 2 3 PCV dose primary series groups shown. At 12 months, each 3 dose group either received a PCV booster or no booster. 2 HBV co-administered with 1 st and 3 rd dose of PCV only 3 GMC following one dose 4 GMC following 2 doses 5 Blood drawn at 24 weeks of age (~6 weeks post-dose 1) 6 DTwP-Hib and OPV co-administered with 1 st dose of PCV only 7 Blood drawn at 6 months pre-dose 3 8 Blood drawn 2 months post-dose 1 9 IPV and Hib co-administered with 1 st and 2 nd dose of PCV only; HBV co-administered with 1 st dose of PCV only 10 Bood drawn 2 months post-dose 2

68 vPPS The relatively inexpensive 23vPPS covered approximately 85-90% of IPD isolates in adults in the US pre-pcv era [254]. The 23vPPS is licensed for use in adults and children 2 years old who have certain underlying conditions which are risk factors for IPD [254]. The characteristics of the immune response to 23vPPS compared with that after polysaccharide protein conjugate vaccines include: a poorer immune response, especially in infants and young children; absent booster responses on repeated administration of the vaccine; minimal maturation of the immune response as indicated by a predominance of low affinity antibody; and no priming. Immunological priming allows a rapid antibody response when an organism is encountered later and may indicate long-term immunity. A 0.5mL dose of 23vPPS contains 25μg of purified capsular polysaccharide from each of the 23 serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 20, 22F, 23F, and 33F). The 23vPPS is administered as a single intramuscular or subcutaneous injection [254] Safety Local reactions are the most common adverse event (AE) following administration of 23vPPS. These reactions usually resolve within 2 days of vaccination and are usually mild [255, 256]. Some studies assessing AEs following re-vaccination reported large local reactions and limb swelling in healthy children and adults vaccinated within 3 or 4 years of first vaccination [ ]. Re-vaccination at an interval of at least 5 years since first vaccination has subsequently been shown to be well tolerated in adults [ ] Immunogenicity Antibody responses to many of the pathogenic pneumococcal serotypes in the 23vPPS are poor in infants, as polysaccharide antigens induce a T-cell independent immune response [263]. Therefore they are poorly immunogenic in those aged < 2 years and fail to induce immunologic memory as a single dose of PPS followed by a booster dose of PPS in children <5 years old does not result in higher antibody levels than a single dose of PPS [264]. Higher antibody concentrations would be expected following re-vaccination if priming had occurred with the first PPS dose. Serotype-specific IgG responses following a single dose of 23vPPS are age and serotype dependent [146, ] with poor responses demonstrated in most infant studies for serogroups 6 [146, ], 19 [146, 263, 264, 267, 268], and 23 [ ], and inconsistent responses in other studies for serotypes/groups 1 [263, 264], 12 [146, 266], 14 [264, 266], and 18 [268]. Serotype 3 can induce antibodies in infants as young as 3 months of age [269]. In the Gambia, antibody concentrations were measured to 6 pneumococcal

69 34 polysaccharides in children before and one month following 23vPPS at the ages of 2, 4, 6, or 9 months or at 5 to 10 years of age [263]. Antibody responses to serotypes 1, 3, and 5 were seen in all age groups but responses to serotypes 19F and 23F were only seen in the older children [263]. Hence responses are particularly poor to those paediatric serotypes that are carried for longer periods, but are better to the more pathogenic serotypes Immunological hyporesponsivesness A particular concern relating to the administration of PPS to young children is the theoretical risk that hyporesponsiveness may occur following re-challenge or subsequent pneumococcal exposure following PPS [189]. These concerns are largely based on 2 observations. First, it has been demonstrated that repeated vaccination with meningococcal C polysaccharide vaccine results in diminished immune response to subsequent doses of the vaccine, even in adults [270, 271]. A study documenting immunologic memory 5 years after meningococcal A/C conjugate vaccination in infancy showed that challenge with the meningococcal polysaccharide or conjugate at 2 years of age displayed evidence of immunologic memory [271]. However subsequent challenge with the polysaccharide vaccine at 5 years of age failed to induce a similar memory response in the polysaccharide vaccinated group. The authors concluded that the initial polysaccharide immunisation at 2 years of age interfered with the immune response to subsequent polysaccharide vaccination [271]. No adverse clinical effects have ever been documented from repeated exposure to the meningococcal polysaccharide vaccine. The clinical significance of this finding is unknown, and meningococcal C polysaccharide vaccine continues to be used throughout the world, especially in countries of the African meningitis belt [272]. In some communities, children are vaccinated yearly. There is no epidemiological evidence of increased susceptibility to meningococcal disease in communities or individuals that have been exposed to repeated vaccination [272]. Secondly, a small pneumococcal vaccine trial undertaken in the Netherlands in children up to 6 years of age with a history of OM randomised to receive a combined PCV and 23vPPS regimen, or a control vaccine found that whilst the primary endpoint (the recurrence rate of OM) was identical in the 2 groups, there was an increase in the total number of OM episodes suffered by the pneumococcal vaccinated group, which just achieved statistical significance at the p=0.05 level [273]. For Australian Indigenous children, the national immunisation schedule consisted of 3 infant doses of PCV followed by a 23vPPS booster at 18 months of age. In a recent retrospective cohort study of Indigenous children in the Northern Territory, Australia, the findings

70 35 suggested an increased risk of hospitalised acute LRTI after pneumococcal vaccination particularly after receipt of the 23vPPS (adjusted hazard ratio after vs no dose, 1.39;95% CI, ; p=0.002) [274]. In contrast, results from Western Australia using a population based data linkage system, found significant reductions in acute LRTI in Indigenous children [275]. However it is unclear what proportion of children had received the 23vPPS. Similarly national data from a national electronic database found a significant decrease in Australian Indigenous childrens hospitalisations for pneumonia [276]. In addition, there is suggestive evidence that the incidence of 19A IPD is less in Indigenous children living in regions where the 23vPPS is given in the second year of life [277]. Immunogenicity studies following re-vaccination with PPS in young children using different valencies and formulations ranging from 5 to 100μg/serotype of PPS have shown inconsistent results including reduced responses to some serotypes following re-vaccination [257, 265]. Conversely, one infant study showed no evidence of hyporesponsiveness on revaccination with PPS [266]. The assays used in these studies were less specific than techniques currently in use. A more recent study in children over 12 months of age who were randomised to receive one dose of the 7-valent pneumococcal polysaccharidemeningococcal outer membrane protein complex conjugate vaccine or 23vPPS at 12, 15, or 18 months of age were further randomised to receive a booster of the conjugate vaccine or 23vPPS 12 months later [186]. Those that had received the conjugate as a primary dose followed by a 23vPPS booster achieved higher antibody concentrations post booster than those that had received the 23vPPS as a primary and booster dose. In the same study, in a non-randomised comparison comparing the immunogenicity of a single primary dose of 23vPPS at 24, 27, or 30 months of age to a booster dose of 23vPPS at 24, 27, or 30 months of age following a priming dose of 23vPPS at 12, 15, or 18 months, lower antibodies were potentially achieved for serotypes 4, 6B, 9V, and 23F. The authors concluded that 23vPPS given at months may induce tolerance to an additional 23vPPS vaccination 12 months later [186]. A small study in which 11 children aged between 2-8 years with recurrent infections who received a second 23vPPS due to an inadequate response to the same vaccine administered 6 months previously, found that the GMC following the second dose compared to those following the first dose appeared to be similar for most serotypes but lower for serotypes 6B and 23F, although these differences were not statistically different [278].

71 36 In vitro studies have suggested that polysaccharides antigens may be able to down regulate B cells [279], and that newly formed antibody via IgG, IgM, or immune complexes can bind to inhibitory Fc receptors and prevent antibody production [280]. Both plasma and memory B cells are stimulated following exposure to PPS. In contrast to T-independent immune responses, priming by either PCV, previous encounter with S. pneumoniae or a cross-reacting antigen prior to 23vPPS vaccination, could stimulate immunologic memory by presentation of polysaccharide-protein conjugate antigens to the immune system (T-dependent) [281]. Given the T-independent nature of PPS antigens, 23vPPS may stimulate the existing pool of memory B cells to differentiate into plasma cells and secrete antibody without replenishment of the memory B cell pool. This has been proposed as one mechanism for the hyporesponsiveness observed following meningococcal polysaccharide vaccine administration [282]. Upon subsequent booster with 23vPPS or a natural infection, immune hyporesponsiveness could be induced as a result of a decreased memory B cell population. The development of immune hyporesponsiveness may also be the result of immune regulation via the establishment of pneumococcal-specific tolerogenic immune responses. Increased expression of the immunosuppressive cytokine IL-10 [264, 283] and suppressor T cell activity may suppress the response to PPS [284]. Recent evidence also suggests a role for CD4 + T-lymphocytes in the immune response to pneumococcal antigens [285]. Studies have demonstrated the importance of co-stimulatory signals (CD40-CD40L) for a robust immune response to pneumococcal antigens and that CD4 + T-lymphocytes can protect mice against pneumococcal colonisation independent of specific antibody. These findings strongly suggest a role for cellular immunity in protection against pneumococcal infection [98, 99, ]. Furthermore it is possible that regulatory T-lymphocytes (Treg) may suppress antibody production and other immune responses in the context of chronic antigen exposure. Hyporesponsiveness induced by Treg has been described during bacterial, viral and parasitic infections with up-regulation of CD4 + CD25 + Treg and IL-10 and TGF-β secretion [284, 289]. Limited data are available on the role of Treg in the attenuation of the immune response to pneumococcal antigens. However a high level of exposure to pneumococci, particularly in early life, could induce Treg activity that suppresses serotype-specific IgG, thereby increasing IPD risk following 23vPPS immunization. The clinical relevance of these immunological findings is not known. There is one case report documenting immunological paralysis for four years to the causative pneumococcal

72 37 serotype in a 9 month old infant who had pneumococcal meningitis, despite demonstrating normal immune responses to other protein and polysaccharide antigens [290] Efficacy Several studies in the 1980s assessed the efficacy of the 8- or 14- valent PPS vaccine in preventing OM in children under 6 years of age [207, 267, ]. Most studies evaluating the impact of PPS immunisation in the absence of additional PCV in infants or children have not shown any impact on pneumococcal disease or carriage [267, 292, 298]. In Australia, a randomised, controlled trial showed no difference (RR 1.01; 95% CI, ) in the risk of acute OM in children <2 years of age and a possible trend towards reduction (RR 0.83; 95% CI, ) in those children >2 years of age [292]. In contrast, a study in Papua New Guinea, where more than 7,000 children aged 6 months to 5 years of age were given either the 14- or 23vPPS in one or 2 doses according to age, there was a non-significant 19% reduction in all-cause mortality, and a 50% reduction in pneumonia mortality (95% CI, 1-75%) [299]. Natural exposure in a population with a high incidence of pneumococcal infections, resulting in regular antigenic stimulation may explain this finding [189]. This study has not been widely accepted, largely due to lack of supportive serological data and questions regarding the study design [300]. The study has not been replicated anywhere, and the vaccine is not used currently for infants anywhere in the developing world, not even Papua New Guinea despite the vaccine showing a significant reduction in mortality (hazard ratio 0.42; 95%CI, ) for children aged between 12 and 24 months [301]. A Finnish study using the 14-valent PPS in infants aged 3 months to 6 years showed significant efficacy of 52% against VT recurrent OM for children <2 years of age if serogroup 6 was excluded [146]. A meta-analysis found no evidence for an overall effect in risk on OM in children <2 years of age who received PPS, but in the pooled analysis of children >24 months of age there was evidence of a vaccine effect (RR 0.78; 95% CI, ) [207] Combined PCV/23vPPS schedules Pneumococcal conjugate vaccines with broader serotype coverage were, until recently, not available. As such some health authorities had decided on or were considering a combination of an infant PCV primary series with a booster of the 23vPPS in the second year of life to address the limited serotype coverage offered by PCV. The theoretical advantage of this approach is to prime with PCV then boost with 23vPPS which is cheaper and may offer some protection against disease due to the serotypes not included in the PCV. These non-pcv serotypes have become more important in IPD now that PCV use is widespread, as the impact of serotype replacement

73 38 leads to a larger proportion of circulating serotypes coming from those serotypes not included in the PCV [19]. Until recently, Australian Indigenous infants routinely receive 3 PCV doses in infancy followed by a booster of the 23vPPS at 18 months of age. This schedule has been found to be immunogenic for most serotypes [302]. For these reasons, and because of its lower cost, the 23vPPS may be the most suitable booster for use in developing countries. There have been several studies involving children in a number of countries using different pneumococcal conjugate formulations and schedules, comparing the immunogenicity of a 23vPPS or PCV booster following a pneumococcal conjugate vaccine primary series. The majority of studies have shown that serotype-specific antibody concentrations are generally higher following 23vPPS than PCV booster [157, 172, ]. The higher response may be due to the higher dose of pneumococcal polysaccharide in the 23vPPS, compared to PCV, enhancing the stimulation of memory B cells or by stimulating a greater number of B cells overall [189]. A randomised controlled trial of infants in Papua New Guinea primed with PCV given at months, months, or no PCV followed by 23vPPS at 9 months of age found a an excellent booster response for the PCV VT, and a 2.8 to 12.4 fold rise in the 3 NVT tested (serotypes 2, 5, and 7F) [303]. A study of vaccine efficacy against acute OM found that a PCV/23vPPS compared to a PCV/PCV schedule had similar results despite higher antibodies generated post PCV/23vPPS [188]. Previous studies have found that the quality of antibody, measured by avidity or OPA, can differ in those that have received 23vPPS or PCV as a booster, however results have been conflicting and therefore inconclusive [154, 157, 186, 304, 305]. Finnish studies have shown the concentration of antibodies required for 50% killing was higher [304] and that the avidity of such antibodies was lower after PCV/23vPPS compared with PCV/PCV [147, 154, 186]. In contrast, another study in Finland using the 11-valent pneumococcal conjugate vaccine showed that OPA was better in the group that received a 23vPPS booster at months than those that had the conjugate booster [305]. A study in Israeli children who received a single dose of the 7-valent pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine followed by either a conjugate or 23vPPS booster, achieved similar opsonic antibody titers in each group for the one serotype tested (6B) [186]. The avidity of antibodies in a limited number of studies on a limited number of serotypes increased following a conjugate booster but not a PPS booster [147, 154] Impact of pneumococcal vaccination on nasopharyngeal carriage Acquisition and carriage of pneumococci is associated with the occurrence of acute OM [11],

74 39 bacteraemia [9], and pneumonia [10]. In developing countries, colonisation rates can be >60% by 2 months of age [15, 16]. An intervention that reduces or delays carriage could result in a decrease in IPD and possibly a decrease in mucosal disease. PPS vaccines do not significantly impact on NP carriage [306]. In contrast, clinical trials using the 5, 7, or 9-valent pneumococcal conjugate vaccines have shown a reduction in VT carriage compared with unvaccinated infants [ ] or toddlers [ ]. However the overall rate of pneumococcal NP carriage has remained essentially unchanged due to serotype replacement from NVT [306, 307, ]. Since the routine introduction of PCV into infant national immunisation schedules, there have been a number of carriage surveys documenting the effect of PCV on pneumococcal NP carriage. Similar to the clinical trials, all studies have found that there has been a reduction in VT carriage [ ]. For NVT, colonisation has increased following vaccination, with serogroups 11 and 15 being commonly reported in many studies [ , 318, 320, 321], and more recently the newly identified serotype 6C has been reported [322]. Moreover, the widespread use of infant PCV in the US has resulted in significant protection of unimmunised individuals [17, 323] presumably mediated by reduced NP carriage interrupting the transmission of pneumococci [17, 18]. In the UK and some Scandinavian countries, a 2 PCV dose schedule in infancy followed by a PCV booster towards the end of the first year of life is routinely given. Little is known about the effect of reduced dose PCV schedules in terms of their impact on carriage and subsequent effect on herd immunity. There is one published randomised controlled trial reporting the effect of reduced dose pneumococcal conjugate vaccine schedules on NP carriage [324]. This study from the Netherlands compared carriage rates following 2 PCV doses at 2 and 4 months of age with a 2+1 schedule at 2, 4, and 11 months of age, and an unvaccinated control group. Both vaccinated groups had significant reductions in VT carriage in the second year of life compared with controls [324]. The booster dose resulted in an earlier further reduction in VT carriage at 18 months compared with no booster dose (24% vs 16%). However by 2 years of age both vaccinated groups had similar VT carriage rates (15% each) [324]. Similarly, in a case-control study Gambian infants, vaccinated with either 3 or 2 doses of a 5-valent pneumococcal conjugate vaccine in infancy followed by 23vPPS at 18 months of age showed a significant reduction in VT carriage compared with unvaccinated matched controls at 2 years of age [306]. An observational study of Portuguese children aged between 12 and 24 months of age in day care centres showed a single PCV dose led to serotype replacement between VT and NVT isolates, both in single and multiple serotype

75 40 carriers, in contrast to the unvaccinated control group where no replacement phenomenon was detected [325]. This was evident at both the population and individual level [325]. In addition, a single dose of PCV decreases VT colonisation as it prevents de novo acquisition and increases NVT colonisation by enhancing NVT unmasking [325]. PCV has had larger than anticipated herd immunity effects on IPD in the unvaccinated elderly and other age groups [19, 52]. However replacement disease, particularly due to serotype 19A, has developed due to capsular switching and clonal expansion [220]. Despite being a significant reduction overall in pneumococcal meningitis in the US, a recent study has shown that the rate of non-pcv serotype meningitis has significantly increased by 60.5% and the proportion of penicillin non-susceptible pneumococcal meningitis isolates increased significantly to pre-vaccine levels [228]. However, serotype replacement may be more of a concern for the control of OM and pneumonia although there is no evidence of this so far. Finnish infants given PCV in a clinical trial showed an increase in NVT OM incidence by 33% although the overall effect was a reduction in disease [161]. In contrast to PCV, studies using PPS have shown no effect on pneumococcal carriage [ ]. One study in the Gambia where 5-valent pneumococcal conjugate vaccinees or Hib vaccinated infants were given 23vPPS at 18 months of age found that the matched controls had signifcantly higher rates of VT carriage than the pneumococcal vaccinated groups [306]. NVT were found more frequently in the pneumococcal vaccinated groups than the control group indicating 23vPPS had no beneficial effect on NVT carriage [306]. In the Netherlands PCV followed by a 23vPPS booster given to children aged between one and 7 years with recurrent acute OM found no beneficial effect from the booster vaccine [273]. A Finnish study of vaccine efficacy against acute OM in infants found that infants given a 3 dose PCV followed by a 23vPPS booster had no further beneficial effects to a PCV booster following a 3 PCV dose primary series a despite higher antibodies generated post the PCV/23vPPS schedule [188]. It is important, therefore, to assess NP carriage and serotype distribution with the introduction of PCV, as the impact of serotype replacement is likely to be dependent on the pattern of serotypes circulating in the community. Moreover, as NP carriage of VT may be an indicator of vaccine efficacy, the combination of this effect with immunogenicity data may help to identify which immunisation schedules are likely to be more effective.

76 Rationale Access to Vaccines Approximately 73% and 69% of infants in sub-saharan Africa and south Asia, respectively, have received 3 doses of diphtheria-tetanus-pertussis vaccine [331]. Deaths from ARI occur mainly in communities with poor health care access [332]. Vaccine provision to children in these countries depends on health care access and financial resources. Children in remote regions often receive incomplete immunisation courses, and doses are usually given later than recommended. At the commencement of this study the price of PCV was approximately US$50 per dose, and as such the recommended schedule of 4 doses, at the time, was unlikely to reach those children at greatest risk. At the commencement of this study there was only one licensed vaccine (Prevenar, formerly Wyeth Vaccines now Pfizer Inc.) in the US and Australia. In 2010 the 10- and 13-valent pneumococcal conjugate vaccines have now reached licensure. Therefore, the present PCV was the only one of its type available at the time this project commenced and its price was likely to remain unaffordable for developing countries for quite some time. Recently novel financing mechanisms for low income countries have been developed called the Advanced Market Commitment and the International Finance Facility for Immunisation. However for lowmiddle income countries (including Fiji), who will not benefit from these financing mechanisms, it is important to investigate new strategies to deliver this vaccine that are safe and effective, but also affordable and that provide flexibility in terms of the number and timing of doses Evaluation of Alternative Pneumococcal Vaccine Schedules is a Research Priority In 2001, a meeting was held at the National Institutes of Health (NIH), Bethesda, Maryland, to determine research priorities to facilitate the use of pneumococcal vaccines in developing countries. The discussions spanned epidemiological, clinical, technical, policy and logistical impediments to the introduction of these vaccines to the world s poorest countries. The recommendations from the meeting included a list of priority research activities, including the need to evaluate alternative regimens of PCV, to provide regimens that are more affordable, provide more flexibility for countries where repeated and timely immunisation visits are atypical, and provide protection in early infancy. The following strategies, amongst others, were identified for evaluation: regimens that include only one or 2 doses of PCV

77 42 (rather than 3 or 4), those that combine PCV and the 23vPPS, and those that include a neonatal dose. The vaccine trial in this thesis addresses the first 2 strategies Immunological Basis to the Vaccine Trial Design Because PCV induces priming, it is possible that it will protect even in the absence of a detectable antibody response. This raises the possibility that fewer doses of PCV than are presently recommended, perhaps even a single dose, may be sufficient to protect from serious IPD. Antibody levels achieved after 3 doses are usually sustained for only a few months, and then decline close to pre-immunisation levels. However a dose of pneumococcal vaccine, either 23vPPS or PCV, administered during the second year of life to children primed with 2 or 3 doses of any of the PCV, generally induces a rapid, 10-fold increase in antibody concentrations to most serotypes, consistent with priming [173, 175, 176, , 333]. 23vPPS has been shown to elicit a booster response following 2 or 3 doses of PCV for the shared 7 PCV serotypes. The additional 16 non-pcv serotypes may offer broader serotype protection. However the immunological safety of 23vPPS booster in young children needs to be evaluated. Vaccine schedules that combine one to 3 doses of PCV with a dose of 23vPPS may lead to later hyporesponsiveness in vaccinated children similar to that found with the meningococcal C polysaccharide vaccine [271, 282]. It may be that this hyporesponsiveness is found in susceptible individuals only, or it may be that it is found with certain serotypes only, or both. Therefore, the Fiji study has been designed to address this question, as well as addressing the immunogenicity and impact on carriage of regimens combining PCV and 23vPPS. For the vaccine study we aimed to find a vaccination strategy for resource poor countries in terms of serotype coverage, flexibility, and affordability. We undertook a Phase II vaccine trial to document the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination regimens combining one, 2, or 3 doses of PCV in infancy. In order to broaden the serotype coverage, the additional benefit of a booster of 23vPPS at 12 months of age was also assessed. To address the theoretical concerns of hyporesponsiveness to 23vPPS following re-challenge, the immunological responses at 17 months of age to a small challenge dose of 20% of 23vPPS (mpps) in infants who had or had not received the 23vPPS at 12 months of age was undertaken.

78 Knowledge to be Gained Pneumococcal infections are the most common cause of morbidity and mortality in children globally. This study has profound implications for the early introduction of pneumococcal vaccines, in particular, to the world s poorest countries. As the current schedule recommends 3 doses in infancy, it is unlikely to be affordable for most developing countries. Evidence from the US suggests that fewer doses are required for protection against IPD, and even modest immunity may be sufficient to prevent children from dying from pneumococcal disease. In addition, many infants in developing countries have episodic access to health care and immunisations. It is important, therefore, to investigate affordable, safe, flexible and effective ways to deliver this vaccine. The need to evaluate alternative regimens of PCV was identified as an important research priority by a recent WHO/ GAVI joint meeting to address impediments to the introduction of these vaccines in developing countries. This study has been deliberately formulated with that need in mind. For the development of the study, we imagined a possible future set of recommendations for the use of pneumococcal vaccines in remote areas with minimal access to health care. Under such circumstances, children presenting during the first 6 months of life could receive one to 3 doses of conjugate, while those presenting later with any experience of conjugate vaccination could receive a dose of 23vPPS Evaluation of Alternative Pneumococcal Schedules in Fiji Fiji is a developing country with a strong immunisation system and an established track record in the introduction of new vaccines. It was likely that Fiji would have been one of the first developing countries to introduce PCV, provided the disease burden is established and an appropriate regimen defined. Hepatitis B vaccine was introduced in Fiji over 10 years ago. Fiji was the first country in the Pacific to have introduced Hib vaccine. Negotiations with Wyeth-Lederle in 1995 resulted in 150,000 free doses of Hib vaccine being donated to the government. The initial Hib campaign commenced in mid and ceased in mid A cluster survey in 1996 found that 79% of children less than 12 months old had completed the primary series. Routine immunisation began again in In early 2009, a coverage survey found the third dose coverage of DTP-Hib/HepB to be >98%. 1.8 Objectives My thesis comprises a series of studies documenting the pneumococcal disease burden and a Phase II pneumococcal vaccine trial in the resource poor setting, Fiji. The overall objective

79 44 was to gather sufficient evidence for the Fiji MoH to decide whether to introduce the pneumococcal vaccination into its national schedule and define an appropriate and affordable vaccination strategy. The objectives were to: 1) Document the prevalence of NP carriage of pneumococci, risk factors for carriage, antimicrobial susceptibility patterns of carried pneumococci, and the serotypes of carried pneumococci in healthy young children in Fiji; 2) Document the burden of IPD and serotype distribution in all ages in Fiji. The primary objective of the vaccine trial was to: 1) Demonstrate non-inferiority in GMC for 11 or more of the 23 23vPPS serotypes, one month post-mpps, in the groups that have or have not received the 12 month 23vPPS. The secondary objectives of the vaccine trial were to: 1) Demonstrate non-inferiority one month post-mpps, between those groups receiving 23vPPS at 12 months and those who do not with respect to the proportion of children: a) in each group with antibody levels 0.35 g/ml to each of the 23 serotypes included in the 23vPPS, by ELISA. b) with OI 8 to each of the 11 serotypes for which OI data were available. 2) Compare the proportion of infants at 18 weeks of age with antibody levels by ELISA, to 7 PCV serotypes 0.35 and 1 g/ml following a primary series of 3 doses of PCV in infancy or an alternative schedule of 0, 1 or 2 doses. 3) Compare the GMC of ELISA antibody at 18 weeks of age following a primary series of 3 doses of PCV in infancy or an alternative schedule of 0, 1 or 2 doses. 4) Compare the GMC for 23 23vPPS serotypes, at 12½ months of age following a booster of 23vPPS at 12 months of age, following a 0, 1, 2, or 3 dose primary series of PCV in infancy. 5) Compare the proportions of children who prior to mpps carry VT and NVT pneumococci in the nasopharynx among children who have received one, 2, or 3 doses of PCV in infancy with or without a booster dose of 23vPPS at 12 months of age.

80 45 Tertiary objectives were to compare: 1) Serotype-specific GMC for 3, 2, 1 or 0 dose PCV groups with or without the 12 month 23vPPS one month following mpps. 2) The median avid antibody levels in those who did or did not receive the 12 month 23vPPS booster for the 23 23vPPS serotypes and had 3, 2, 1 or 0 dose PCV groups for the 7 PCV serotypes. 3) The proportions of children carrying a VT and a NVT for the 7 PCV serotypes for NP swabs taken at 6, 9, 12, and 17 months of age for the different groups Change to the Original Objectives The FiPP vaccine trial commenced recruitment in October 2004 with support from NIAID and the National Health and Medical Research Council (NHMRC). However, recruitment was suspended by NIAID in February 2005 after 228 infants had been recruited. Due to an administrative oversight within NIAID the trial had not passed through the required internal review within NIH, so recruitment was suspended pending that review. According to the original protocol, infants were randomised to receive one, 2 or 3 doses of PCV in early infancy and a booster dose of 23vPPS at 6 or 9 months. Two control groups were also recruited to receive either 23vPPS at 9 months or no pneumococcal vaccine prior to 15 months of age. Outcomes to be measured were antibody levels (ELISA, OPA, and avidity), pneumococcal NP carriage and response to mpps at 15 months of age. Following the review by NIAID this design was rejected on the grounds that they believe there was insufficient safety data to support the use of 23vPPS vaccine in infants under 12 months of age. Specifically fears had been raised that administration of one or more of the serotypes included in the 23vPPS vaccine may lead to diminished response to some pneumococcal antigens on subsequent re-exposure, potentially leading to an increased risk of disease. This had not been specifically addressed in earlier studies. This concern, raised by the Technical Review Group convened by NIAID, was based on recent studies of meningococcal C polysaccharide vaccine, in both children and adults, which indicate that this widely used vaccine leads to lower responses to the same vaccine on revaccination [271]. The NIAID advisors expressed concerns that this phenomenon may also occur with one or several of the serotypes included in the 23vPPS vaccine, either when this vaccine is given alone or following earlier PCV. The protective efficacy of this vaccine against pneumonia mortality when delivered to infants in Papua New Guinea provides reassurance

81 46 in this regard [299, 301]. On the other hand, in a recent study from the Netherlands in which older children with recurrent OM were vaccinated with a combined PCV and 23vPPS regimen, while the proportion of vaccinated children suffering subsequent OM was identical to controls, the total number of episodes suffered by vaccinees appeared to be greater [273]. Following the NIAID review, we were instructed not to give 23vPPS to infants at either 6 or 9 months of age and instead to redesign the study to evaluate the immunological safety of 23vPPS at 12 months. We were also advised not to proceed with the original sample size of 1,060 but redesign a smaller study. As a result of these concerns, the design had changed to evaluate the immunological safety of 23vPPS at the same time as evaluating alternative pneumococcal vaccination regimens. Therefore the redesigned FiPP had the primary objective of testing the following hypothesis: 1) To demonstrate non-inferiority one month post-mpps, between those groups receiving 23vPPS at 12 months and those who do not with respect to the proportion of children with OI 8 to each of the 11 serotypes for which OI data are available. Furthermore, during the course of the trial and following discussion with the Data Safety and Monitoring Board (DSMB) in November 2007, the definition of hyporeponsiveness changed from what was originally defined in the protocol. The original definition of hyporesponsiveness has been changed to non-responsiveness. As such the primary objective was as stated in section Objectives Potential Risks There was a small risk of local reactions following 23vPPS administration to children who have been previously immunised with PCV. In addition, there was a risk that some children may have reduced responses to some pneumococcal antigens following receipt of 23vPPS. As was discussed previously, this was based on experience with meningococcal C vaccine, which represents the only model of hyporesponsiveness following polysaccharide administration in humans [271]. There is no evidence that this phenomenon has led to any adverse effects in the millions of individuals who receive meningococcal C polysaccharide each year [272]. While it was possible that this study may uncover hyporesponsiveness to one or more pneumococcal polysaccharide serotypes, it was unlikely that this would be of clinical significance. None of the studies that have been conducted up to now have documented any adverse effects in children as young as 3 months of age receiving 23vPPS

82 47 [146, 292, 299, 334, 335]. The combination of PCV and 23vPPS being employed in this study was similar to that used as a regimen for high risk Indigenous infants in Australia until the 10-valant vaccine was introduced in the third quarter of 2009 with a 4 dose schedule. Pain was likely to be felt by the infants with each venipuncture. There was a small risk of bruising and redness at the venipuncture site. Rarely infections may occur as a result of venipuncture Known Potential Benefits The potential benefits of vaccinating individual infants for protection against pneumococcal disease outweighs the potential risks. There is extensive experience with PCV in trials in developed and developing countries, and in all cases it has been shown to be safe and efficacious [52, 336]. All children in this trial will receive pneumococcal immunisation that is likely to be effective. Those groups that receive 0 or 1 dose of PCV in infancy will receive a dose of PCV at the end of the trial, at 2 years of age, when a single dose is known to be effective. 1.9 Hypothesis The original primary hypothesis for analysis was that 23vPPS at 12 months will not lead to a greater proportion of non-responsiveness to mpps at 18 months for any of the 11 serotypes for which functional analyses are available. This was to be tested by determining whether, for each of the 11 serotypes, if the proportion of children showing non-responsiveness was 15% greater (in absolute terms) in the 23vPPS group than in the group that had not received the 12 month 23vPPS. Differences in proportions and their confidence intervals were to be calculated using the standard normal approximation, in view of the large sample size. The test was to be the standard test of non-inferiority of proportions, single sided (as recommended by ICH guidelines for non-inferiority trials), with a significance level of 5%. To determine non-inferiority we were to calculate single-sided 90% confidence intervals for the difference. The proportion of child who had or had not received the 12 month 23vPPS with serotype-specific OI 8 will be declared non-significant if the lower bound of these confidence intervals excludes -10%. It was anticipated that within a child, serotype responses would not be independent of each other. In other words, it was likely that a child who was non-responsive to one serotype would be non-responsive to a number of serotypes. It was assumed that for any given serotype, 30% of children were expected to be

83 48 non-responsive, and 30% of children will be non-responsive to at least half of the serotypes (4 or more).

84 49 2 MATERIALS AND METHODS 2.1 Setting The Republic of Fiji islands lies within the South Pacific Ocean. It comprises two large islands, Viti Levu and Vanua Levu and more than 300 smaller islands (Figure 2). Fiji s population of 827,900 is primarily comprised of Indigenous Fijians (57%), who are predominantly Melanesian, and Indo-Fijians (38%) who are of Indian ethnicity (2007 Census). Over 75% of the population lives on Viti Levu which has two medical divisions, the Western and Central divisions. The population in the Central Medical division is 371,850. The total population of Suva, the capital, is 75,225 and the population of Nausori, its neighbouring satellite, town is 24,630. Together they contain approximately one third of the Central Division s population and from here the participants of this study were recruited. The literacy rate is approximately 90% with English as the official language, but Fijian and English are taught in schools. Hindi is the third language. Fiji is classified as a low middle income country. The poverty rate is 35% and the GDP per head of population in 2006 was approximately FJ $5474. (Source: Fiji Islands Bureau of Statistics, 2008). The infant mortality rate is 18.4 per 1,000 live births (Fiji Ministry of Health Annual Report, 2007). The <5 child mortality rate is 22.4 per 1,000 live births. In 2007 there were 19,298 live births (Fiji Ministry of Health Annual Report, 2007) of which approximately one third were in the Suva and Nausori area.

85 50 Figure 2: Map of the Republic of the Fiji islands showing the 4 medical divisions Source: Fiji Islands Bureau of Statistics, 2008