A Randomized Clinical Trial of the Immunogenicity of 7-Valent Pneumococcal Conjugate Vaccine Compared to 23-Valent Polysaccharide Vaccine in Frail, Hospitalized Elderly

Background Elderly people do not mount strong immune responses to vaccines. We compared 23-valent capsular polysaccharide (23vPPV) alone versus 7-valent conjugate (PCV7) vaccine followed by 23vPPV 6 months later in hospitalized elderly. Methods Participants were randomized to receive 23vPPV or PCV7-23vPPV. Antibodies against serotypes 3, 4, 6A, 6B, 9V, 14, 18C, 19A, 19F, 23F were measured by enzyme-linked immunosorbent (ELISA) and opsonophagocytic (OPA) assays at baseline, 6 months and 12 months. Results Of 312 recruited, between 40% and 72% of subjects had undetectable OPA titres at baseline. After one dose, PCV7 recipients had significantly higher responses to serotypes 9V (both assays) and 23F (OPA only), and 23vPPV recipients had significantly higher responses to serotype 3 (ELISA), 19F and 19A (OPA only). In subjects with undetectable OPA titres at baseline, a proportionately greater rise in OPA titre (P<0.01) was seen for all serotypes after both vaccines. The GMT ratio of OPA was significantly higher at 12 months in the PCV7-23vPPV group for serotypes 6A, 9V, 18C and 23F. OPA titre levels for these serotypes increased moderately after 6 months, whereas immunity waned in the 23vPPV only arm. Conclusion We did not show overwhelming benefit of one vaccine over the other. Low baseline immunity does not preclude a robust immune response, reiterating the importance of vaccinating the frail elderly. A schedule of PCV7-23vPPV prevents waning of antibody, suggesting that both vaccines could be useful in the elderly. Follow up studies are needed to determine persistence of immunity. Trial Registration The Australian Clinical Trials Registry ACTRN12607000387426


SCIENTIFIC PROTOCOL
with that of the over, without a ctogenicity and at PCV7, given alone or followed by a dose of PPV, does not confer any additional benefit to that offered by PPV alone for the same serotypes of S. pneumoniae in t common cause y. Australia has ed as a national lia recommends ococcal vaccine those at greatest ailable since the end of 2000, but is is vaccine uses linicals trials of otects 93.9% of isease (IPD). Our study aims to look at the efficacy of this new vaccine, currently only registered for children, in lation who are at highest risk for pneumococcal disease -hospitalised re their immune implications for eumonia, and a cteraemia, otitis e in the over 60 le, people with sm, as there are penicillin and multidrug resistant pneumococcal 6 eventive strategies d a report titled ked in order of on public health d those against abetes, multiple The next twenty years will see an increase in the Australian population of over 65 year-olds from 12.5% to 18%, and a doubling of those over 85 years old. Pneumonia in the frail elderly will place increasing demands on our health care system, and better prevention will not only relieve much suffering, but has the potential to result in significant cost savings. The National Health and Medical Research Council (NHMRC) recommends that all adults aged 65 years and over should be immunised with 23-valent polysaccharide pneumococcal vaccine (PPV) at least once. 8 Younger persons who have medical risk factors are also recommended for immunisation. PPV has been available in Australia for decades, and has been shown to be cost-effective in these groups. 9 This Aims: To compare impact of the 7-valent pneumococcal conjugate vaccine (PCV7) 23-valent polysaccharide vaccine (PPV) in hospitalised adults aged 60 years and past history of vaccination with polysaccharide vaccine on immunogencity, rea nasopharyngeal colonisation with Streptococcus pneumoniae. Null hypothesis: Th hospitalised elderly adults.

Simple Description:
The bacteria pneumococcus (also known as streptococcus pneumoniae) is the mos of pneumonia in the community, and a major cause of illness and death in the elderl an ageing population. The health and wellbeing of the elderly has been identifi priority. The National Health and Medical Research Council (NHMRC) of Austra that adults aged 65 years and over should be immunised with polysaccharide pneum (PPV). PPV has been available for many years in Australia, but is least effective in risk, the sick and the elderly.
A new pneumococcal conjugate vaccine (PCV-7) has been av currently indicated only for children, because it has never been tested in adults. Th different technology, and is conjugated to a protein to make it more effective. C PCV7 have largely been limited to children aged 0-4 years, and have shown it pr children under 2 years of age against invasive pneumococcal d the sub-group of the popu elderly. We will vaccinate hospitalised elderly people with PCV or PPV and compa response to the two different vaccines. If PCV is more effective than PPV, this has the development and use of conjuagated pneumococcal vaccines for adults.

Background/Literature Review
Streptococcus pneumoniae is the most common cause of community acquired pn major cause of morbidity and mortality in the elderly. 1 It also causes meningitis, ba media, and other infections. The mortality rate for invasive pneumococcal diseas age group ranges from 5-57%, with higher mortality in immunosuppressed peop underlying chronic diseases and those of advanced age. 2-5 It is a complex organi over 80 known serotypes. In addition, rates of disease are increasing in Australia and other industrialised countries, so that pr such as vaccination are increasingly important. The Institute of Medicine release "Vaccines for the 21st Century" in 2000, where hypothetical vaccines were ran desirability based on burden of disease, associated costs and likely positive impact and society in general. The most desirable vaccines for human health include pneumococcal disease (along with vaccines against cytomegalovirus, influenza, di sclerosis, rheumatoid arthritis and group B streptococcus). 7 vaccine confers protection against 23 pneumococcal serotypes, which cover t invasive disease in adults. However, a meta-analysis of clinical trials has shown effective in younger, immunocompetent persons. 10 The antibody response to PPV in in people with pre-existing medical conditions is less predictable, and observatio suggested reduced or no efficacy in he majority of that it is most the elderly and nal studies have very elderly or immunocompromised.
The dilemma associated with the use of PPV is that it is least effective in those at greatest risk of pneumococcal ide to enhance occal vaccines. e response to ccines has the ty and to confer ensed 7 valent young children, uded in the 23s are less likely 1,900 invasive itted for routine serotyping; 69.2% and 76.6% and 96.1% and polysaccharide requency) were olates were on 3 and 22F. Given the likelihood of suboptimal efficacy rage of PCV-7 ion of immune immunogenicity ent and use of s been licensed in Australia since the end of 2000. Clinicals trials of PCV7 are limited to isease (IPD) of ent in antibiotic xpected to have n replacement is riage and herd ernationally on conjugate pneumococcal genic than PPV. ococcal disease US looking at aged >65 years (personal correspondence, Dr Mary Ramsay Health Protection Agency UK and Dr Cynthia Whitney Centres of Disease Control Atlanta ). These groups agree that there is still a need to study the use of conjugate vaccine in sick, elderly populations where high disease burden and sub-optimal PPV responses may justify the use of a substantially more costly vaccine. The unique contribution of our study will determine the immunogenicity of PCV7 in a vulnerable elderly population with known high incidence of IPD. It is impractical to measure efficacy in such a group in a clinical trial so proxy measures of efficacy using immunologic surrrogates is necessary. With increasing demands being placed on our health care system by an ageing population, more effective prevention of IPD in this group could result in significant cost savings. 18 the 11,12 disease and its complications, the sick elderly. 10 Recently, conjugate technology, whereby a protein is linked to a polysacchar immunogenicity and confer immuonologic memory, has been applied to pneumoc The primary aim is to enable young children to mount a protective immun pneumococcal polysaccharides, but the improved immunogenicity of these va potential to induce improved responses in older persons with impaired immuni protection of longer duration through memory responses. The currently lic pneumococcal conjugate vaccine (PCV-7) targets the serotypes most common in namely 4, 6B, 7V, 9V, 14, 18C, 19V and 23 F. These serotypes are all also incl valent PPV, along with 16 other serotypes. Isolates causing invasive disease in adult to be among the serotypes included in PCV-7. During 2001-2002, in Australia, isolates from non-Indigenous adults were subm belonged to serotypes and serogroups, respectively, included in the 7-valent vaccine 96.6% belonged to the 23 serotypes and groups, respectively included in the vaccine. The most common serotypes isolated from adults with IPD (in order of f 14,4,9V,23F,6B,3,19F,18C,22F,6A,19A,7F,9N,8,11A,1,12F. 13 Thus the top 10 is cross-reacted with 7 valent serotypes except data for PPV in sick, elderly populations, it is possible that the lesser serotype cove may be more than compensated for by increased immunogenicity and induct memory. Regardless of the ultimate clinical value of PCV-7, if improved compared to PPV can be demonstrated, this has implications for further developm conjugated pneumococcal vaccines of higher valency.
PCV7 ha children aged 0-4 years, have shown 94% efficacy against invasive pneumococcal d vaccine serotypes in children under 2 years of age. 14 The serotypes most preval resistant pneumococci are also those most common in young children, so it can be e a disproportionate impact on reduction of antibiotic resistant pneumococci. 15 Strai also an issue of interest, as is the effect of vaccination on nasopharyngeal car immunity.
Currently, there are very limited 16 published data int vaccines in adult populations. In adult renal transplant patients it is more immuno 17 With its increased immunogencity, it may be a candidate for prevention of pneum in vulnerable adults.Two immunogenicity studies are underway in the UK and the scheduling options and immune responses in healthy adults

Methods:
Design: A randomised, clinical trial of PCV7 + PPV compared to 23 valent polysaccharide vaccine ic, cardiology or vaccine, will be st have complex, multi-system pathology, as d by self-report l, Sydney. The cute emergency ssions a year of have multiple ent and patients ating syndrome re or if they are lity, because of the commonest ns, septicaemia s per year. The e and syncope, rn Sydney Area nts have agreed s patients with complex rheumatological and autoimmune disorders, most of whom are on immunosuppressive y NHMRC. All The Cardiology heart disease or rdiologists have estimation of sponse rates between groups with 95% confidence intervals, as detailed in an excessively rotype pre-post en be compared between schedules and between age groups using t-tests. For the purposes of the sample size calculations the estimated t in an grou taken as 0.55, which dy (M Ramsay, Approximately 150 subjects are required in each arm, allowing for failure to complete the protocol in 15-20% of each study arm in this vulnerable population. To allow for unexpected additional follow up problems, we will aim to recruit 200 in each arm as follows: 1. PCV7, followed 6 months later by PPV, no past vaccination with PPV 200 2. PPV, no past vaccination with PPV 200 The case fatality rate for admitted inpatients is 15-18%, reflecting the severity of illness and comorbidity in this group. We expect an 80% participation rate, consistent with recruitment to other (PPV) alone in hospitalised elderly patients.
Subjects/eligibility: Any patient 60 and over years of age admitted under the geriatr rheumatology unit at Westmead hospital, who has not received pneumococcal eligible. To be admitted under this unit patients mu described below. Patients with a history of pneumococcal vaccination (as determine and validation by general practitioner records) will be ineligible.
Setting: The Geritaric, Cardiology and Rheumatology units of Westmead Hospita Westmead Department of Geriatric Medicine has seven consultants and offers an a geriatric medicine service 24 hours a day. There are between 2,000 and 2,500 admi 1,500 individual patients (1,300 inpatients and 200 day-patients, some of which admissions). Westmead Hospital does not have a general internal medicine departm are generally admitted to geriatric medicine if they present with a decompens (delirium, falls, incontinence or immobility), if they usually live in institutional ca 60 and over years of age and are not considered appropriate for any other specia multiple pathology, especially cognitive impairment and dementia. Pneumonia is single reason for admission to geriatric medicine (7%), with respiratory infectio and exacerbations of chronic obstructive airway diseases accounting for 300 case commonest diagnostic categories are respiratory infection, heart failure, collaps stroke, urinary sepsis and septicaemia [data from the Decision Support Unit, Weste health Service for financial year 2002/3]. There are 200 deaths a year. All consulta to their patients being approached for the study. The rheumatology unit admit treatments. They are therefore in the category indicated for pneumococcal vaccine b consultants in the unit have agreed to their patients being approached for the study. unit is the busiest unit in the hospital. Most patients are admitted with ischaemic cardiac failure. This is also an indication for pneumococcal vaccination. All ca agreed to their patients being approached for the study.
Sample size: A sample size of 132 subjects per vaccine group will enable reactogenicity and re Table C. The study size will be sufficient to identify a poorly immunogenic or reactogenic vaccine. For each individual the change in log-22FA levels for each se vaccination will be calculated. The average change will th standard deviation for the change in log10-22FA levels wi h y p is was the average standard deviation observed across serotypes in a UK pilot stu personal communication). trials by this Unit. We estimated that in NSW, where pneumococcal vaccine is re not funded, about 30% of the population aged >65 years will be already commended but nated, based on program.. 19 The for the elderly gram for adults eriatric patients r the study. This is the justification for extending the age vaccine, and are ve consent. We itive impairment, as it is important not to exclude this eed to have a carer able to make the s (see below). Guardianship Board approval will be required if patients do not have capacity to consent. This is defined as: e to the consent d the applicants c medicine has extensive experience with consent issues in geriatric clinical trials. 20, 21 The Department is currently 10 year history ecturer to create experience and daily basis, and r records. This is necessary because a previous Australian hospital-based study in which CIA was involved established that iring validation cination will be possible effects ion. This is also hen randomised . Equal numbers men than men. ation (on-site by ill be recorded prior to treatment allocation of a er covering the ossible to blind rm receives two vaccinations, 6 months apart. The study nurse and CI's will be blinded to the first dose and for assessment of reactogenicity measurement), but the differing presentation of PPV and PCV7 might lead to trial staff being able to differentiate between vaccines, even if the labels are removed. Allocation will be need to be known at 5 months to organise the follow up doss of PPV for subjects who received PCV-7. Laboratory testing will be blinded.
Vaccination procedure: The study nurse will perform immunisation, sample collection and subject follow-up. The vaccination will be deferred if the subject's temperature is above 38C or they have an acute illness. Day of vaccination will be Day 1. Vaccines will be administered by intramuscular vacci Victorian data comparing vaccination rates pre-and post funding of the >65 years study was conceived and funded before the new free pneumococcal vaccine program came into being. Early recruitment after the funded pneumococcal vaccination pro aged >65 years, which commenced in January 2005, shows that >80% of admitted g are vaccinated, making them ineligible fo range to 60-64 years, which then includes patients who are not eligible for free likely to have baseline vaccination rates of <30%. Consent: Written informed consent will be obtained from patients who are able to gi will also include people with dementia and cogn group of frail elderly patients. However, such patients will n relevant reactogenicity measure • being capable of understanding the general nature and effect of the trial • being able to retain the information • being able to communicate this information to the researchers. Even when patients are deemed to not have capacity, they can still often contribut process. Appropriately short and simple trial materials will help in this regard, an (RIL) have experience in this area. 20 The Westmead Department of Geriatri recruiting patients in four randomised controlled trials of stroke treatment and has a of successful trial recruitment. The recent appointment of a Professor and Senior L a new academic department of geriatric medicine has provided the necessary resources to expand clinical research.
Recruitment and enrolment: The trial nurse will identify all new admissions on a will validate vaccination status from hospital and general practitione reported vaccination status of hospitalised elderly patients was often unreliable, requ by general practice records. 19 If consent is given by the patient and/or guardian, vac performed at the end of the hospital stay, to ensure the optimum balance between on immunogenicity of acute illness and the high costs of out-of -hospital vaccinat relevant as it is likely that such a policy would be most suitable for implementation.
Randomisation: Patients will be recruited, consented, baseline data collected and t by a personal computer which records, in a secure fashion, all patients randomised of men and women will be recruited, bearing in mind that the unit admits more wo We will balance randomisation by age (continuous variable) and gender. Randomis computer) will be secure as baseline details w numbered vaccine vial. The vaccine vials will be blinded to the patient (by a stick label). Patients will be initially blinded for the 1st vaccine dose, but it will not be p patients in the long term, since one arm receives a single vaccine and the other a injection in the arm. The nurse will write the subject's initials and date of admi adhesive label bearing the subject's study number and stick this onto the empty vac Pneumococcal vaccine will be given in the non-dominant arm. No other va administered concomitantly. Subjects will be observed for 15 minutes after vacc nistration on an cine container. ccines will be ination for any immediate adverse reaction. Standard immunisation practices will be followed and appropriate e the subject or ometer, pen and to complete the ne on day 1 and ascertained at 3 it). The causal events and vaccination will be categorised by the investigators, as one graded as mild, rapeutic Goods 20mls of blood t the expressed ill not vaccination but will preclude participation in the study. Serum samples will be taken by venepuncture before, and 12 months after vaccination to determine vaccine specific antibody responses. (see elo wab will be taken at study entry and at the 12 month follow up visit. The trial nurse will be trained in proper nasopharyngeal collection methods.

outcomes:
ryngeal colonisation with S.pneumoniae specific IgG avidity after each dose to assess immunological mem ntibody assays using opsonophagocytosis (to be done at 12 months only) a) geometric mean fold rises (with 95% confidence intervals) of the vaccine serotype titre for each For the PCV7 arm, immunogenicity to PCV7 alone will be measured at 6 months, after which a dose of PPV will be given. At 12 months, the immunogenicity of this PCV7-PPV schedule will be measured. For the PPV arm, immuogenicity to a single dose of PPV will be measured at 6 and 12 months.
We will measure IgG antibody to each of the PCV7 serotypes by ELISA, which has been long established in CIB's laboratory, with appropriate World Health Organization reference standards. We will also measure seroresponse to the 23-valent PPV vaccine serotypes which are most prevalent but not contained in PCV7 (types 3 and 22).
precautions for any anaphylactic reactions will be taken.
Assessment and notification of reactogenicity: The vaccine study nurse will giv carer a health diary labelled with their study number together with a digital therm ruler. The nurse will provide instructions on how to use the thermometer and how health diary. Study nurses will complete an adverse event questionnaire by telepho day 7. Any medical consultations, medications taken and hospitalisations will be months, 6 months (follow up visit), 9 months and 12 months (follow up vis relationship between serious of the following: related; probable; possible; remote or unrelated. Events will be moderate or severe. If appropriate, such events will be notified to the The Administation via the "Blue Card" Collection and testing of samples: Blood samples: At least 10mls but no more than will be taken. No more than two attempts at venepuncture will be made withou verbal consent of the subject to continue. Failure to obtain a blood sample w then at 6 and b w) Nasopharyngleal samples: A nasopharyngeal s Main 1. Immunogenicity: (see below for details) 2.
Nasopha Secondary outcome measures -using a subset of 30% of study participants 1.
antibody response to the carrier protein pre and post vaccination 1. Immunogenicity: a) specific IgG antibody in all participants. b) the proportion of subjects achieving a 1.5 fold rise in antibody pneumococcal serotype As it is possible that the potency of such antibody if present may be reduced, functio established with well-validated methodology in CIBs labortatory -will also be perfo two international studies currently underway ( nal assays-also rmed. The only comparing PCV7 and PPV in healthy adults) are n literature, due ral are different ne is prevention immunogenicity clinical data. An example is meningocococcal C conjugate vaccine, implemented as a national program in the UK, Australia and the Netherlands on the nicity to the two vaccine schedules by telephone follow up, and if a) the proportions of subjects with significant (>2.5cm) local reactions to study vaccines for ation. and 7 days of 2 months postthe serotypes carers at 7 days after vaccination to be followed up at 6 months and 12 ion in hospital ryngeal specimen or PPV) llow up Serology: both immunogenicity studies, without clinical endpoints. This is a longstanding problem, which is recognised in the pneumococcal vaccinatio to the rare nature of clinical events. 22 It should be noted that vaccine trials in gene from therapeutic trials, because the outcomes are rare and the effect of the vacci rather than treatment. In fact, increasingly, vaccines are registered on the basis of data alone, in the absence of which has been basis of immunogenicity data alone.

Reactogenicity:
We will measure reactoge required by a visit from the trial nurse. This is defined as seven days following vaccin b) the proportions of subjects with pyrexias (>38oC ) within 3 days immunisation.

Nasopharyngeal colonisation:
A nasopharyngeal swab will be collected from all participants at baseline and 1 vaccination, to ascertain nasopharyngeal colonisation with pneumococcus and involved.

Follow up:
The study nurse will telephone all patients or their ask about reactogenicity to the vaccine. Patients will then months after baseline sera are collected. At these milestones, the study nurse will visit patients in their place of residence to conduct follow up. Baseline (date of vaccination): clinical data collect Serum collection (for serology) Nasopha Vaccination (PCV& 7 days: Telephone fo 6 months: Home visit Clinical data collection Serum collection Vaccination with 23vPPv for PCV arm only. 12 months: Home visit Clinical data collection Serum collection Nasopharyngeal specimen Laboratory methods: Serum will be collected from patients at baseline, 6 months and 12 months after vaccination. As part of a CRC-Vaccine Technology funded project Dr Sullivan's group has recently completed a study of pneumococcal-specific antibodies in a blood donor population. This involved the screening of over 800 hundred blood donors for any pneumococcal antibodies by ELISA and a further characterisation of the serotype-specific antibodies in a sub-group (~6% of the study population) with an anti-pneumococcal titre greater than a standard pneumococcal reference serum (prepared from a pool of individuals vaccinated against pneumococcus). In addition, functional antibodies (defined by a flow cytometric opsonophagocytosis assay) were also studied against three common disease-causing serotypes (6B, 9V and 23F). This study is in the final stages of data analysis and it is anticipated that a paper will be submitted shortly. T standard assays for the characterisation of pneumo hus, both gold coccal antibodies (ie ELISA and the flow opsonophagocytois assays) are well established in our laboratory.
Pneumococcal antibody ELISA. Antibody levels to each of the seven individual (4 and 23F) polysaccharide serotypes that are common to the PPV and PCV vaccines using an enzyme-linked immunosorbent assay (ELISA) according to the cons ,6B,9V,14,18C will be measured s protocols. 23,24 fied pneumococcal tion from 22F 23,24 l dilutions (this pared using C-PS following: (a) entration and ts as required.
added to the 37°C for 5 hrs in a shed with 300 ng an automated cycles. The assays bumin (BSA; 200 µl/well, 2 h nd incubated for 2 elevant QC and C-PSare added shed 5 times and i-human IgG-alkaline phosphatase conjugate added and the plates again ed substrate (pan additional 2 ped by the addition erence filter 23,24 . LISA program ensu All serum samples (both pre and post vaccination) will be preabsorbed with puri cell wall polysacharride (C-PS) (Serum Statens Institute) and a capsular prepara and antibody concentrations will be determined for each sample using three seria will be done for each of the seven serotypes) and read off a standard curve pre absorbed 89-SF reference serum. Assay conditions will be optimised 23,24 for the concentration of individual polysaccharides used to coat the ELISA plates, (b) conc absorption times for C-PS and 22F and (c) additional individual ELISA reagen In brief, each antigen is diluted in 0.05 M sodium bicarbonate buffer (pH 9.6), then wells of microtiter plates (Maxisorp; Nunc, Roskild, Denmark) and incubated at humidified environment (or at 4°C overnight). After each step, the plates are wa µl/well of phosphate-buffered saline (PBS; pH 7.2) containing 0.1% Tween 20 usi plate washer (Wellcozyme 812 SW2) programmed for a 30-s soak and five wash are performed at room temperature. PBS-Tween 20 containing 2% bovine serum al Sigma Chemical Co., St. Louis, Mo) is used for blocking non-specific binding ( incubation) and for further dilutions. All serum samples are tested in duplicate a h.. Serial dilutions of the C-PS and 22F absorbed patient serum samples and the r standard reference standards (note reference serum 89-SF is only absorbed against and the plates incubated for a further 2 hrs at room temperature. The plates are wa a dilution of a goat ant incubated for another 2 hrs at room temperature. After a further 5 washes a dilut nitrophenyl phosphate) is added and the plates incubated at room temperature for hrs ( or working substrate solution,20 min incubation). The reaction is then stop of 50 ul of 3M NaOH and optical density measured at 405 nm using a 690 nm ref Optical densities will then be converted to antibody concentrations using the CDC E (available via the CDC, 25 ). Flow cytometric opsonophagocytic assay. The opsonophagocytic assay will be perf consensus protocols 26, 27 with minor adjustments. Briefly, the seven serotypes 4,6B, 9V, 14, 18C and 23F (obtained from ATCC) are plated on 5% sheep blood ag Australia) and grown over night in a 5% CO 2 atmosphere at 37°C. Isola haemolysis are then inoculated into 5 ml Todd-Hewitt broth with 0.5% yeast extrac Dickinson) and incubated without shaking for 3 h. Bacterial cultures are grow highly encapsulated pneumococci. The bacteria are then pelleted by centrifugati min at room ormed using of S. pneumoniae ar (Oxoid, ted colonies displaying α t (Beckton n three times to obtain on (800 × g for 10 NaHCO 3 [pH 8.0]). nimidyl ester Sigma]) and incubating the mixture for 1 h without shaking at 37 C in 5% CO 2 . Following addition of 1 ml of 2% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA), cells are fixed over night at 37°C without shaking. Non-viability of the labelled bacteria is confirmed by culturing 100 µl of bacterial suspension on a blood agar plate as described before. Testing of pneumococcal carriage: entiation of these cells into granul achieved using the same medium supplemented with 100 mM N, N-dimethylfo a period of up to 7 days with a cell density of 4 x 10 5 -6 x 10 5 . Differentiated HL harvested by centrifugation (160 × g for 10 min) and then washed twice in 15 ml o opsonophagocytosis buffer. Cells are counted and the concentration adjusted to 2.5 Prior to flow cytometric opsonophagocytic analysis, serum samples are heated to inactivate any endogenous complement activity. The flow cytometric opsono done in two stages. Initially, the samples are screened for the baseline antibody (lowest dilution) in duplicate. In brief, 20 µl of neat serum is transferred tubes (Falcon) coated in Sigmacoat (Sigma), then 40 µl of bacterial suspension ( added and incubated in a shaking water bath at 37°C for 30 min. Then, 20µl baby complement (Csix, USA) is added to each tube except the HL-60 cell control (to w opsonophagocytosis buffer is added) and incubated at 37°C for 15 min. 80µl (2.0 × washed, differentiated HL-60 cells are then added and incubated for a further 30 the end of this incubation, 160 µl ice cold 0.9% NaCl/0.02% EDTA solution to stop phagocytosis. Samples are then kept on ice prior to analysis. Titre tubes µ and 100 l of 0.5% trypan blue (Trace Scientific) added to quench fluorescence of bacteria. Then titre tubes are placed inside polystyrene tubes for flow cytometric an controls are included in each assay: (i) HL-60 control containing only cells and ba complement control containing all reagents except antibody source, a sample (serum from an adult vaccinated with the 23-valent pneumococcal vaccin Samples will be analysed using a Beckman Coulter EPICS Elite EPS flow cy ed of 5000 gated HL-60 granulocytes was analysed per tube. FAM-SE is excit nm, and the FAM-SE signals of the gated viable HL-60 cells measured at 530 of background fluorescence is measured using HL-60 cell controls and consisted o autofluorescence of HL-60 cells. An analysis region is placed above the ce peak and included 98% of the population. A second analysis region is used to d percentage of HL-60 cells with a fluorescence greater than the control cells for e Serum samples with a maximum phagocytic uptake of ≥ 30% will be analysed furt opsonophagocytic titre of these samples will be evaluated in the second stage. S dilutions of test sera, beginning with neat, are made in the round-bottom microtiter plates coated in Sigmacoat (Sigma), then transferred to polypropylene titre tubes, and the flow cy opsonophagocytic assay done as described above. The opsonophagocytic titre is d reciprocal of the highest dilution that gives ≥ 50% with a maximum phagocytic uptake of < 30% are considered negative and will be re a titre of 4. The presence of significant functional antibody is defined as a titre ≥ 32. Competitive inhibition will be carried out using homologous and heterologous polys Serum samples and a positive control sample (1:16 lowest dilution tested) will be te opsonophagocytic antibodies to the 7 pneumococcal serotypes (4,6B, 9V, 14 18C an preabsorption for 30 min at room temperature with equal volume of homologous or Nasophayrngeal swabs will be collected using a calcium alginate on wire swab. Swabs will be inoculated directly on to 5% horse blood agar containing gentamicin 5mg/L, by rolling it over ¼ of the plate's surface. In the laboratory, the inoculum will be streaked on to all 4 quadrants using a sterile loop. Plates will be incubated for 18-24 hours (37oC, in 5% CO2) and then examined for the presence of alpha-haemolytic colonies resembling pneumococci. Species will be confirmed by optochin sensitivity and bile solubility. Growth will be graded as scanty (<25 colonies on quadrant 1 only); 1+ (>25 colonies in quadrant 1 and <25 colonies in quadrant 2); 2+ (>25 colonies in quadrant 2 and <25 colonies in quadrant 3); 3+ ( quadrant 3 and <25 colonies in quadrant 4) 4+ (>25 colonies in quadrant 4) ( Several colonies resembling pneumococci fr >25 colonies in O'Brien, 2003). om each plate will be subcultured and serotyped, using bed molecular method (Kong, 2003).
to reduce the nt (eg serology, ferent endpoints for correlations Software, Inc., ill be expressed ch polysaccharide. Antibody titres s will be made and Chi-squared on between opsonophagocytic titre and antibody concentration will be calculated using linear correlation coefficient.
l polysacharide NHMRC. It is nly 30% of the ted. Study subjects will be unvaccinated. All subjects in the PCV7 arm (ie receiving the new vaccine), will also receive PPV one month later. Therefore, no study subject will unvaccinated at vaccine has any ions, but there is enced after the elderly people tudy may show t pneumococcal ties in vaccinee are at greatest e an increase in , and a doubling accination is an important preventive health measure in this age group. The existing polysacharide pneumococcal disease is least effective in this population. A protein conjugate pneumoccocal vaccine is more immunogenic in children under 2 years (who do not respond to PPV), but only covers 7 serotypes. Protein conjugate pneumococcal vaccines are likely to be more immunogenic in adults and should confer immunologic memory, relevant to the need for repeated doses in adults. No studies have examined nasopharyngeal carriage of pneumococcus following PCV in adults. Our study will allow comparison of response to PCV7 alone to PPV at 6 months, as well as to priming with PCV7 followed by PPV at 12 months. This trial has the potential to increase the local awareness of pneumococcal vaccination. Participation in the trial is likely to a recently descri Statistical analysis. Intention to treat analysis will be done. We will use appropriate corrections probability required to accept statistical significance. Each immunological endpoi functional antibodies) will be analysed separately, and correlations between the dif will also measured. Within the sub-group that are to be studied, we will look between serology and functional antibody for a number of specific serotypes. Statistical analyses will be carried out using InStat software, version 3 (GraphPad San Diego, CA) and standardised against the CDC ELISA program. The results w as geometric mean concentrations (GMC) of IgG antibody to ea will be log transformed prior to analysis and comparison of titres between group using analysis of variance and regression analysis if required. Fishers exact test test will be used where appropriate to compare proportions of seroconverters. Correlati Comparisons of local and systemic reactions to routine and study vaccines will be m squared and Fishers exact test where appropriate. 95% confidence intervals will also References ETHICAL ANALYSIS Potential risks: The major ethical implication of this study is that pneumococca vaccine (PPV) is recommended for all adults aged 65 years and over by the estimated that in NSW, where this vaccine is recommended but not funded, that o target group are vaccina be denied the recommended vaccine, and all subjects stand to benefit, as they are the time of enrolment, and would not have received vaccination otherwise. Neither serious side effects. The vaccine being trialled, PCV7, may cause minor local react no evidence that the incidence of these will be any greater than those experi recommended vaccine, PPV.
Potential benefits: The major benefit is that the study will result in unvaccinated receiving pneumococcal vaccine, which is recommended for this age group. The s that in this highly vulnerable population, PCV7 offers better protection agains disease than PPV. Pneumococcal diseases is one of the highest ranked priori preventable causes of human morbidity and mortality globally. Sick, elderly peopl risk of pneumococcal disease and its complications. The next twenty years will se the proportion of over 65 year olds from 12.5% to 18% of the Australian population of those over 85 years old. Healthy ageing is a national research priority, and v benefit the individual who will receive a recommended treatment which they had not previously been offered.

Proposed date of commencement: May 2005
: three years sent forms have fted and are attached to this application. Those who agree to participate in the study will be informed of their results and what those results means in relation to their immunity pneumococcal ants will be encouraged to contact the study investigators if they have any

Review of Progress:
l meet fortnightly to review the study progress. Monthly data reviews ly significantly icity data, the study will be prematurely terminated. easure reactogenicity to the two vaccine schedules by telephone follow up, and if dy vaccines for nd 7 days of immunisation.
pyrexias will be treated symptomatically with paracetamol where required. versely affected rticipation, then the Children's The study will terminate 12 months after the last patient is recruited. Patients will be followed up at tones, the study does not involve e 12 months of vided by the study investigators at the Westmead Hospital geriatric unit. Feedback from this study to participating ococcal disease.
Data will be kept on password protected computer files for the duration of the research. Hard copies of the data will be kept in a locked filing cabinet. Only the study investigators will have access to the computer and paper files. The computer files will be kept long term in a password protected database. All identifying data will be coded and de-identified for participant's privacy.

Will data be collected from a Federal Government agency?
No data will be obtained from any government agency for use in this study.

Care of Participants
This study will not affect the patients's relationship with the hospital. Patient con been dra disease. Particip concerns.
The study investigators wil and basic analyses will be performed. If one vaccine appears to be statistical favourable over the other, based on immunogen

Management of Adverse Events
We will m required by a visit from the trial nurse. This is defined as a) the proportions of subjects with significant (>2.5cm) local reactions to stu seven days following vaccination. b) the proportions of subjects with pyrexias (>38oC ) within 3 days a Local reactions and Subjects will also be encouraged to call NCIRS if they think that they have been ad by participation in this trial. If the patient becomes ill or is injured as a result of pa reasonable costs of medical treatment will be paid by Westmead hospital and Hospital at Westmead.

Winding up procedures
7 days, 6 months and 12 months after baseline sera are collected. At these miles nurse will visit patients in their place of residence to conduct follow up. This study any longer term, on-going care of participants beyond the 12 months. During th follow up, any required care directly as a consequence of the study will be pro patients will be in terms of their immunity to pneum

Access to data
Data will be kept on password protected computer files for the duration of t computer files will be kept long term in a password protected database. Hard copi questionnaire will be kept in a locked filin he research. The es of the g cabinet. The paper files will be retained for five years and will be disposed of using a document shredder.