Observational study to investigate vertically acquired passive immunity in babies of mothers vaccinated against H1N1v during pregnancy

Effect of maternal vaccination and acquired (vertical/passive) immunity in children

Influenza vaccination in pregnancy offers benefit to the mother by reducing the risk of infection and the resultant complications. It has also been established that immunisation in pregnancy with trivalent, unadjuvanted, seasonal influenza vaccine does provide vertical immunity to the child through the cord blood. 17–19,22,24 However, the clinical impact of the finding is less clear, with some studies indicating benefit and others not. 16–19 The same is not true of a monovalent, two-dose schedule, new variant influenza vaccine with or without adjuvant, where the scientific evidence for vertical transmission of passive immunity, although likely, has not yet been established.

Rationale for the proposed study

Given the current risk profile of the emergent pandemic virus, it could be assumed that pregnant women would readily choose to be vaccinated. However, perception and response to threats and assessment of risk do not necessarily align in terms of human behaviour. Research has suggested that people tend to overestimate the likelihood and impact of rare events and underestimate for more common situations. 27,28 The public response to the measles, mumps and rubella (MMR) vaccination scare, and its possible link to autism and Crohn’s disease, is an example. Despite substantial and sound evidence to the contrary, many parents chose to refuse MMR vaccination for their children on the basis of a theoretical association that has by most scientists and policy-makers been dismissed, therefore exposing them to the risk of serious disease from measles, mumps or rubella. Another example of a similar response is that to whooping cough vaccine in the 1970s. Whether the low take-up of seasonal influenza vaccine in pregnant women in the USA is due to similar anxiety is uncertain; however, policy-makers will be concerned to ensure maximum uptake of the H1N1 swine flu vaccination. Therefore, evidence to support the approach may help encourage women to come forward for immunisation. If data were available which revealed that vaccination of pregnant women appeared to confer meaningful protection against swine influenza to their babies after birth, this would enable messages to pregnant women of potential vaccinees to be shifted from ‘evidence that you are likely to benefit and no evidence that your baby will be harmed’ to ‘evidence that you are likely to benefit and further evidence that your baby will also benefit’.

The researchers propose to assess the immunity conferred to infants of mothers who have been vaccinated against H1N1 swine influenza by obtaining umbilical cord blood samples at delivery and submitting them for serological analysis, comparing with unvaccinated mothers in the same birth cohort.

Additionally, the study also plans to assess the efficacy of vaccination of the mother at prevention influenza/respiratory illness in the infant. This will be by following the child up for a period after birth and taking nasal swabs to look for influenza/other respiratory viruses and comparing the rate of infection in those of mothers who were vaccinated versus those who were not.

Purpose

To improve policy implementation by providing clarity on the degree of protection transferred from the vaccinated mother to infant and to enable clinicians to provide pregnant patients with accurate information with which they can make an informed decision over whether to accept immunisation or not, and to allow public health messages to be strengthened.

FIGURE 1.

Observational study to investigate vertically acquired passive immunity in babies of mothers vaccinated against H1N1v (swine influenza) during pregnancy.

Objectives

Primary objective To determine the proportion of babies who have acquired passive immunity to A/H1N1v (swine influenza) born to mothers who accept vaccination as part of the national vaccination programme whilst pregnant (during the second and/or third trimesters) against the novel A/H1N1 swine influenza virus compared with unvaccinated mothers.

Secondary objective To record and investigate influenza-like illness during winter 2009–10 and also to flag for long-term follow-up of babies born to mothers who take part in the study.

Study configuration

Observational cohort study across three centres [Queen’s Medical Centre and City Hospital, Nottingham, and Leicester Royal Infirmary (University Hospital Leicester)].

Full randomisation is not possible within this study, as the participants will/will not have previously been vaccinated as part of the national swine influenza vaccination programme.

Recruitment will be at delivery. Although the researchers would prefer to recruit prior to delivery, they recognise that this has real practical difficulties, especially ensuring that those consented antenatally are sampled at delivery. Therefore, it is considered that recruitment will have to occur at partition. For these reasons it is probable that the majority of participants will be women at term; however, those in the earlier stages of pregnancy (second or third trimester) and presenting in premature labour will be eligible for recruitment.

For the secondary objective (protective effectiveness of the vertical immunity provided to the baby from the maternal H1N1 swine vaccine against acquisition of A/H1N1 swine), subject to the availability of additional funding, the mother will be contacted after birth by the research midwife and provided with nasal swabs to obtain samples if the child becomes symptomatic with a influenza-like illness. The mother/parents will be provided with an information sheet including diagrams on how to do this and will also be trained by demonstration in taking these. With the participant’s consent, intermittent follow-up calls will be made to the mother over the period to 31 March 2010 to reinforce and remind her about swabbing if the child becomes ill. (If further funding can be obtained this may be extended to 31 July 2010.)

With maternal consent a flag will be applied to the ONS record of the mother and child (from both the vaccinated and unvaccinated groups).

Statistical analysis will be undertaken in two stages as a minimum: firstly when all the cord samples (anticipated December 2009) have been obtained and secondly when the nasal swabbing sampling is complete (spring 2010).

Randomisation and blinding

As such, because the exposed participants to be used in the study will be those who have already been vaccinated against H1N1 swine and the unexposed participants controls will be those who have not, it is not possible to randomise at this primary selection level. However, it will be important to avoid the introduction of bias at this stage. It is possible that those accepting vaccination (exposed) as part of the national programme may represent a different group from those who do not/who have not yet been offered it (unexposed). For this reason, data at the point of recruitment on age, ethnicity, socioeconomic status, gravida, parity and pre-existing medical history will be sought to allow further analysis/enable potential confounders to be adjusted for. This should help to reduce the effect of any biases.

For obvious reasons the subjects cannot be blinded, as they will know if they have been vaccinated or not and, therefore, likewise it will not be possible to blind the midwife recruiting/delivering the baby and taking the cord blood sample for the primary objective. Additionally for the secondary objective it will not be possible to blind the mother for nasal sampling. However, the cord blood serological analysis and nasal swab PCR determination will be blinded to the laboratory by use of unique code allocation to sampling. The results will then have to be unblinded at this point in order to allow statistical analysis. The Study Statistician will not have any direct contact will any of the participants.

Unique codes will be applied to the sample request forms and media with no other identifying data so that the laboratory will be blinded. The code will be linked to a separate participant study record that will be sealed and stored securely and will be accessible only to the study team after receipt of the result in order to allow linkage and statistical analysis. (See maintenance of codes below for further detail.)

Enrolment will be coordinated by research midwives at the respective delivery units as previously listed above, with support from the usual care teams (midwife/obstetrician).

Unblinding

Unblinding of the cord sampling/nasal swab PCR results will occur once they are received to allow linkage to the participant study record, entry into the database and subsequent statistical analysis. The researchers will statistically analyse the results as the investigation proceeds to provide interim analysis.

Trial Steering Committee composition

The TSC will comprise the Chief (Principal) Investigator, the co-investigators and the Study Statistician (as listed earlier). Additionally, the study group will endeavour to engage a research consultant neonatologist and general practitioner (GP) to the TSC.

Trial Steering Committee terms of reference

1. To monitor and supervise the progress of the study towards its interim and overall objectives.

2. To review at regular intervals relevant information from other sources (e.g. related studies).

3. To consider the recommendations of the Ethics Committee.

4. In the light of 1, 2 and 3 above, to inform the NIHR Board on the progress of the investigation.

5. To advise the NIHR on publicity and the presentation of all aspects of the study.

End of the study

The end of the study will be date based and is to be 31 March 2009 (subject to additional funding caveat as above).

Recruitment

The study setting will be at the antenatal clinics/obstetric units of the three hospitals listed previously (Queen’s Medical Centre and City Hospital, Nottingham, and Leicester Royal Infirmary). These are all teaching hospitals. They have been chosen for the following reasons:

• large delivery numbers per year (in excess of 20,000 deliveries per year combined)

• established research bases for obstetrics and influenza/infectious diseases at each site

• close proximity to the researchers.

Clearly, this study is focusing on pregnant women and their babies, so all other groups will be excluded, i.e. men, older persons and older children. Pregnant women will be eligible to take part regardless of ethnicity or socioeconomic status.

Participants will be recruited from the delivery units of the above hospitals. The potential participant will be approached by a member of the patient’s usual care midwifery team for possible recruitment to the study. Information about the study will be on display in relevant clinical areas and advertised as widely as possible so that pregnant women are aware of the research before recruitment. If the patient is interested in partaking then the usual care midwife in the relevant unit will then go through the details of the investigation with the potential participant of all aspects pertaining to participation in the study, including obtaining consent.

All of the maternity units have experience of recruiting women to studies during labour and this has been found to be an acceptable time to obtain consent. One of the key midwifery roles in labour is to act as the woman’s advocate. The usual care midwives are therefore very well qualified to decide when or if it is appropriate to approach a woman during her labour, depending on her level of distress and the possibility of clinical complications. The maternity unit will be paid for each cord sample taken but the money will not be paid directly to the usual care midwives – it will be used to improve general staff facilities in the unit.

The researchers have considered carefully whether pregnant women should be recruited antenatally, prior to parturition. However, logistically, it will be not possible to ensure that at delivery the samples of consented mothers are taken. For this reason, it is viewed that women will have to be enrolled at delivery.

Research midwives at each site will help to facilitate participation and coordinate the practical aspects of specimen collection.

If needed, the usual hospital interpreter and translator services will be available to assist with discussion of the study. The participant information sheets and consent forms will be available printed in other languages as far as is reasonably practical. It will be explained to the potential participant that entry into the study is entirely voluntary and that her treatment and care will not be affected by her decision. It will also be explained that she can withdraw at any time. In the event of her withdrawal it will be explained that the data collected so far will be retained.

Inclusion criteria

All pregnant women in the second and third trimester will be eligible to participate in the study, if they present in labour; however, due to the short timescales required by the study commissioners to obtain results, the largest group recruited is likely to be those at term.

Women will be included regardless of age (up to 39 years 364 days), social class, ethnicity, gravida and parity status, past and current medical history (including current medications), ethnicity mode of delivery and pregnancy outcome (live/stillbirth). However, all of these parameters will be noted for each participant to allow further analysis later.

The researchers have thought carefully about whether to exclude women who had underlying health conditions that might predispose them towards being vaccinated, as it is possible that their condition may alter the development of an appropriate immune response (e.g. if on immunosuppressants/renal impairment). However, the conclusion reached was that the information from this group was particularly important and therefore should be included, but to avoid the confounder effects adjustment at analysis may be required.

Exclusion criteria

Primary objective:

The main exclusion criterion is pregnant women in the first trimester/women delivering before the age of fetal viability (23 weeks and 6 days’ gestation).

Other exclusion criteria will be:

• incapacity to provide informed consent for participation refusal

• prisoners

• inability to take cord blood samples (e.g. cord blood needed for other clinical purpose, so none available for the study)

• involvement in another study entailing clinical interventions

• women who do not routinely live in the East Midlands

• assisted conception

• age > 40 years.

Secondary objective:

• Inability to measure the outcome – this may be relevant if the woman is not normally resident in the UK or is moving abroad shortly after the child’s birth or is homeless and of no fixed abode. Compliance here may also be an issue. It may be that the possible participant may have such difficulty understanding the requirements of the secondary objective that it will not be practically possible to include her and her child.

• Refusal by the participant to agree to both parts of the study.

Expected duration of participant participation

Mothers, up to 6 months in total, and their babies, up to 5 months, depending on date of delivery (unless funding allows extension).

Removal of participants

Participants may have to be removed for the secondary objective if they cannot be contacted; however, this would only be accepted as a loss to follow-up if two reminder letters fail to re-engage the participant.

During the enrolment period, where pregnant mothers are being recruited for cord blood sampling at delivery and subsequent follow-up of the baby, if the participant withdraws consent the researchers will seek to replace the lost individual if the number recruited to that date is insufficient to meet the power requirements of the study. After the enrolment period, if a mother subsequently declines to partake in nasal sampling then it will not be possible to replace that individual, as there will be no corresponding cord blood sample to provide comparison (exposed/unexposed to vaccine).

Abrupt termination from the study will not have safety implications for the participant.

Participants may be withdrawn from the study either at their own request or at the discretion of the Principal Investigator.

Informed consent

All participants will provide valid, written, informed consent. The Informed Consent Form will be signed and dated by the participant before she enters the study. The usual care midwife or team/research midwife will explain the details of the study and provide a Participant Information Sheet, ensuring that the participant has sufficient time to consider participating or not before obtaining consent if she wishes to partake. The midwife will answer any questions that the participant has concerning study participation. Consent will be obtained from the potential participant at the point of enrolment. Informed consent will be obtained from each participant before she undergoes any interventions (including history-taking and cord blood sampling) related to the study. One copy of this will be kept by the participant, one will be kept by the Investigator (and stored with the participant’s study record), and a third will be retained in the patient’s hospital records.

Consent will be obtained through face-to-face discussion with the potential participant.

The consent form will be kept with the case report form. Should there be a need later to amend the study (unlikely unless the study commissioner requests a subsequent modification) then each participant will be approached individually and reconsented by the research midwife or other member of the research team. This will be by written informed consent, which will again be stored in the same way as the original consent.

Explicit consent will be sought for both the primary and secondary objective participation, and the use and retention of the relevant data.

At the consent stage it will be made clear to the participant that the cord sample will be destroyed at the end of the study and will not be used for other scientific investigations. Participants will also be advised at this point that the sample will also not be available for subsequent use for clinical reasons, such as paternity determination or stem cell treatment of the baby if later required (as the sample collection and storage would not be appropriate to meet this need) and therefore if cord blood storage for this purpose was desired it would be for the participant to discuss and agree with the usual care team.

Compliance

Compliance with meeting the primary objective is unlikely to be a problem from a patient perspective. The research midwife coordinating on site will be based on the maternity unit as much as practicable in order to optimise enrolment and sample collection.

During the secondary objective phase (nasal swabbing), compliance will be promoted through the regular telephone contact of the participants and direct enquiry at that time whether the infant has had any respiratory infection in the intervening period and if so if they have been swabbed. Where there is a mismatch, the history of a respiratory infection without a swab having been taken will be noted and the mother reminded on the importance of obtaining a swab under these circumstances.

Criteria for terminating the study

The investigation may cease at any of the proposed centres if the adherence to the study protocol is unacceptable or the trust requests it. However, these are not anticipated to be likely and steps to correct problems would be identified first.

There are unlikely to be any specific safety issues with the cord blood sampling that would lead to complete study termination. (See below in the section on the transport and storage of the tissues re. the safe handling of the samples.) Likewise, this is also unlikely with the nasal swab sampling component, as there is established precedent from other studies for this method. Additionally, the mother/parents will be taught how to sample safely.

Possible external reasons for ceasing the whole investigation might include major and sustained business continuity problems at the relevant hospitals/investigating units, either as a direct consequence of the influenza pandemic or other issues. New information from another study source globally may render the need for the investigation unnecessary, unforeseen fund withdrawal and serology or PCR analysis problem may also lead to the investigation being terminated early. Additionally, if the vaccination programme were terminated this would prevent acquisition of vaccinated mothers and would necessitate ceasing the investigation. If, however, nationally, a safety issue with the vaccine were raised, it is likely that the study would need to be expanded – subject to additional funding availability.

Cord blood samples

Cord blood samples will be collected in appropriate sample containers. These will be subsequently collected by the research midwife on each site and packaged according to usual handling standards into appropriate containers designed for the safe transport of biological specimens (safe potential biohazard sample handling). Each sample and the accompanying request form will be labelled with the participant’s unique code only. As described elsewhere in this document, the unique code can be linked to the participant’s study record to allow linkage of the results but maintain anonymisation until then. Once packaged the samples will be transported (by post) to the Health Protection Agency (HPA) Centre for Infections, London, for analysis.

Nasal swab samples

Nasal dry swab samples will be collected in appropriate sample containers. These will be subsequently packaged into appropriate containers designed for the safe transport of biological specimens by the participant (having been previously shown how to do this). Each sample and the accompanying request form will be labelled with the participant’s unique code only. As described elsewhere in this document, the unique code can be linked to the participant’s study record to allow linkage of the results but maintain anonymisation until then. The samples will be transported (by post) to the Leicester Royal Infirmary, Clinical Microbiology Department, for suspension into viral transport medium and subsequent molecular analysis.

At the conclusion of the study, the cord blood and nasal swab samples will be destroyed.

Cord blood serology

All cord blood serology samples will be analysed (by MN and HI) with the NIBRG121 virus [reverse genetics virus based on A/California/7/2009 (vH1N1) and A/Puerto Rico/8/34]. Samples collected at each study site will be centrifuged and separated into two aliquots. Samples will be tested in parallel.

The MN will be performed in 96-well format according to previously described protocols and standard operating procedures (SOPs) developed with the Respiratory Virus Unit (RVU), HPA Centre for Infections.

Elimination of complement (e.g. from fetal calf serum in culture medium) will be achieved by incubation of study sera and appropriate quality control sera (provided and chosen according to test virus by RVU, usually serum of ferret, sheep or human, with/without neutralisation activity) at + 56 °C/30 minutes. This step will be performed simultaneously for all study samples and control sera.

The MN analysis with the NIBRG121 virus will be performed as follows: a six-step twofold dilution series (covering titres 20–640) will be set up for each of the samples and control sera. After addition of a pretitred virus [usually around 100 × median tissue culture infectious dose (TCID₅₀) per well or 0.1-1 virus particle per cell] neutralisation will be performed by incubation of the virus/serum mixture at room temperature for 1 hour. After neutralisation, a suspension of Madin–Darby Canine Kidney (MDCK) cells will be added and the plates will be incubated for 16 hours at 37 °C in a CO₂ incubator. The remaining infectivity of virus after neutralisation is determined in an EIA format using a monoclonal antibody to detect expression of viral nucleoprotein. The amount of nucleoprotein expression is determined photometrically [optical density (OD) reading = OD₄₅₀] using a plate reader.

An OD reading for each dilution step for each sample will be used to calculate the titre. The titre will be reported as the reciprocal dilution at which 50% of the virus is neutralised (e.g. titre of 100). The MN analysis will be performed in duplicate (in separate runs on 2 days) for each sample. The two titres for each sample must not differ by more than a twofold serial dilution. In cases, where samples do not fall within this limit, a third analysis is performed and the two closest titres (which must be within a twofold serial dilution) will be reported.

The principle of the HI test is based on the ability of specific anti-influenza antibodies to inhibit haemagglutination of red blood cells (RBCs) by influenza virus haemagglutinin. The sera to be tested have to be previously treated to eliminate the non-specific inhibitors and the anti-species haemagglutinins. The experiment will be performed in accordance to protocols and SOPs established by RVU.

Elimination of non-specific inhibitors will be achieved by incubation of unknown serum samples and quality control sera (serum of ferret or human immunised with influenza virus) with neuraminidase [receptor-destroying enzyme (RDE) II: 18 hours/+ 36 °C followed by heat inactivation 1 hour/+ 56 °C]. All samples – sera pre-vaccination, post vaccination and controls – will be prepared simultaneously.

For the HI analysis with the NIBRG121 virus, samples and controls will be titrated in an eight-step twofold dilution series (covering titres 8–1024) and incubated with the haemagglutination antigen suspension (previously titrated to adjust the dilution at 4 haemagglutination units/25 µl; 50% end point). The haemagglutination antigen is not added to the well dedicated to the RDE quality control.

The mixture is incubated for 1 hour at room temperature and 25 µl of the 0.5% RBC suspension (turkey blood) are added. The reaction is left for half an hour at room temperature before reading.

The serum titre is equal to the highest reciprocal dilution, which induces a complete inhibition of haemagglutination. The titre of each quality control serum is close to the previously assigned value (within one serial twofold dilution limits). The RBC controls (RBC suspension without antigen) and the RDE controls do not produce any agglutination. Each serum sample is titrated in duplicate and individual titres will be reported (two for each sample). These must not differ by more than a twofold serial dilution. In cases, where samples do not fall within this limit, a third analysis is performed and the two closest titres (which must be within a twofold serial dilution) will be reported.

Nasal swab PCR

Nucleic acid extraction and real-time PCR (RT-PCR) will be performed by research staff at the Molecular Diagnostics Laboratory of the Leicester Royal Infirmary Microbiology Department, according to the UK National Standard Methods VSOP 25 (RT quadriplex PCR for the detection of influenza) and VSOP 29 [swine-lineage influenza A (H1)-specific fast RT-PCR]. Equipment available for automated nucleic acid extraction includes the Corbett Xtractor Gene and the Qiagen Qiasymphony. RT-PCR analysis will be performed using the Corbett Rotor-Gene. The Leicester Microbiology Department is under the Pathology Directorate of the University Hospitals of Leicester/Leicester Royal Infirmary. Regular participation in performance evaluations, such as those of the National External Quality Assessment Service (NEQAS) and HPA, are the SOP to maintain laboratory accreditation.

Methods

The overall programme manager, Dr Richard Puleston, Associate Professor of Health Protection at the University of Nottingham, will be responsible for the evaluation and analysis of the findings of the study. He will be assisted in this task by the statistical expertise provided by Dr Puja Myles and other statistical experts from the same unit. stata 11 will be the primary statistical analysis software, with additional use of Microsoft excel (Microsoft Corporation, Seattle, WA, USA) and other graphing/statistical software as appropriate.

It is likely the researchers will use the following approaches.

Sample size and justification

The researchers have used a conservative power calculation based upon an estimated 20% seroconversion rate in unvaccinated women; in reality it may be closer to 10% as the cumulative clinical attack rate in the UK is still very low (probably < 2%) and so the true serological attack rate is probably < 4%. In addition underlying immunity in the age group 15–44 years (women of child-bearing age) is considered to be low. We have similarly estimated conservatively that seroconversion in vaccinated women will be 50%; in fact it is more likely to be 70% after two doses. Thus a power calculation based on 20% versus 50% is very conservative, and a more optimistic comparison would be 10% versus 70%.

Based on 20% versus 50%, with 80% statistical power and 5% significance (two-tailed statistics), 38 subjects per group are required – total 76. However, we anticipate that two-thirds of women will accept vaccine, so the ratio of unvaccinated–vaccinated subjects will be 0.5; allowing for this imbalance a total study size of 89 subjects (59 vaccinated 30 unvaccinated) would be needed. Assuming a total of 89 subjects and more optimistic estimates of 10% versus 70%, the study would have 100% power to detect such a difference. To allow for possible losses during analysis or inadequate specimens, the researchers plan to recruit a maximum of 100 study subjects.

Assessment of efficacy

Primary efficacy end points will as has previously been indicated be based on the internationally recognised standard for immunity to influenza. Measures of seroconversion as specified by the EMEA–CHMP will be used. These criteria are formally accepted to be measures of seroconversion consistent with clinical protection against seasonal influenza in adults, and are used routinely for testing the immunogenicity of influenza vaccines in adults (HI titre ≥ 40 or single radial haemolysis > 25 mm²). MN tests may be used to assess responses to pandemic influenza but there are no recognised correlates of protection. Subjects achieving MN titres of ≥ 40 are may be considered as an immunogenicity end point. The extent to which these criteria are relevant to pandemic vaccines and to babies could both be debated, but at present there are no widely accepted alternative measures and these criteria are being used for the licensure of pandemic vaccines. They are therefore appropriate for this study.

Laboratory-confirmed influenza in symptomatic neonates will be measured by detection of H1N1 swine by validated PCR from clinical nasal samples.

The efficacy parameter will be the difference in means for both the primary and secondary efficacy end points.

Procedures for missing, unused and spurious data

Missing data may fall into two categories: direct results from the primary and secondary objectives or indirect supporting information, such as date of birth or GP details.

Where the former is missing, every effort will be taken to track down what might be the problem, such as sample not actually taken through to result not being communicated. Where resolvable, the data will then be collated with the other findings. Where data items appear spurious, for example an implausible serological conversion level, clarification will be sought with the testing laboratory to ascertain its veracity and, if necessary, request requantification, although the laboratory will have its own quality control procedures that should detect issues of this nature before reporting.

Where supporting data are missing the research midwives will endeavour to complete the data item either directly from the participant (e.g. number of children under 5 years in the household) or, if more appropriate (e.g. details of the delivery), from the clinical record.

Where data items remain missing despite these efforts, they will be treated as missing and will be noted as such in the analysis.

Definition of populations analysed

Interim analysis set, primary objective Analysis will be ongoing through the study. The relevance of the results as the study progresses will be based on the probability of obtaining such results for the number known.

Interim analysis set, secondary objective This will occur only if the national influenza activity on 31 January 2010 is above the normal baseline for the time of year.

Full analysis set, primary objective All individuals for whom a cord blood sample has been received at the close of recruitment and their subsequent delivery.

Full analysis set, secondary objective All infants for whom a nasal swab has been received by the termination date of the study, 31 March (July – if extended) 2010, or the end of the pandemic, whichever is the sooner.

Efficacy will be assessed on the interim and full analysis set. Ineligible participants will be excluded before recruitment. However, if a participant becomes ineligible through withdrawal of consent then the research record for that person will be marked as such and noted at the time of analysis.

Reporting of adverse events

Participants will be asked to contact the study site immediately in the event of any actual or perceived SAE. All adverse events will be recorded and closely monitored until resolution, stabilisation or until it has been shown that the study intervention is not the cause. The Principal (Chief) Investigator shall be informed immediately of any SAEs and shall determine seriousness and causality in conjunction with any treating medical practitioners.

Participant removal from the study due to adverse events

Any participant who experiences an adverse event may be withdrawn from the study at the discretion of the Principal Investigator.

Ethics committee and regulatory approvals

The study will not be initiated before the protocol, Informed Consent Forms and participant and general information sheets have received approval/favourable opinion from the Research Ethics Committee (REC), and the respective NHS Research and Development (R&D) department. Should a protocol amendment be made that requires REC approval, the changes in the protocol will not be instituted until the amendment and revised Informed Consent Forms and participant and GP information sheets (if appropriate) have been reviewed and received approval/favourable opinion from the REC and R&D departments. A protocol amendment intended to eliminate an apparent immediate hazard to participants may be implemented immediately, providing that R&D and the REC are notified as soon as possible and an approval is requested. Minor protocol amendments only for logistical or administrative changes may be implemented immediately, and the REC will be informed.

The study will be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki, 1996, and the DH Research Governance Framework for Health and Social Care, 2005.

Informed consent and participant information

The process for obtaining participant informed consent or assent and parent/guardian informed consent will be in accordance with the REC guidance, and good clinical practice (GCP) and any other regulatory requirements that might be introduced. The investigator or their nominee (usual/research midwife or obstetrician) and the participant or other legally authorised representative shall both sign and date the Informed Consent Form before the person can participate in the study.

The participant will receive a copy of the signed and dated forms and the original will be retained in the Study Master File. A second copy will be filed in the participant’s medical notes, and a signed and dated note made in the notes that informed consent was obtained for the investigation.

The decision regarding participation in the study is entirely voluntary. The investigator or their nominee shall emphasise to them that consent regarding study participation may be withdrawn at any time without penalty or affecting the quality or quantity of their future medical care, or loss of benefits to which the participant is otherwise entitled. No study-specific interventions will be done before informed consent has been obtained.

The Principal Investigator will inform the participant of any relevant information that becomes available during the course of the study, and will discuss with them, whether they wish to continue with the study. If applicable they will be asked to sign revised consent forms.

If the Informed Consent Form is amended during the study, the investigator shall follow all applicable regulatory requirements pertaining to approval of the amended Informed Consent Form by the REC and use of the amended form (including for ongoing participants).

Records

Data protection

All study staff and investigators will endeavour to protect the rights of the study’s participants to privacy and informed consent, and will adhere to the Data Protection Act, 1998. The CRF will only collect the minimum required information for the purposes of the investigation. CRFs will be held securely, in a locked room or locked cupboard or cabinet. Access to the information will be limited to the study staff and investigators and relevant regulatory authorities (see above). Computer-held data, including the study database, will be held securely and password protected. All data will be stored on a secure dedicated server. Access will be restricted by user identifiers and passwords (encrypted using a one-way encryption method).

Information about the study in the participant’s medical records/hospital notes will be treated confidentially in the same way as all other confidential medical information.

Electronic data will be backed up every 24 hours to both local and remote media in encrypted format.

Insurance and indemnity

Insurance and indemnity for study participants and study staff is covered within the NHS indemnity arrangements for clinical negligence claims in the NHS, issued under cover of Health Service Guidelines (96)48. There are no special compensation arrangements, but study participants may have recourse through the NHS complaints procedures.

The University of Nottingham has taken out an insurance policy to provide indemnity in the event of a successful litigious claim for proven non-negligent harm.

Study conduct

The study conduct will be subject to systems audit of the Study Master File for inclusion of essential documents, permissions to conduct the study, Study Delegation Log, curricula vitae of investigative staff and training received, local document control procedures, consent procedures and recruitment logs, adherence to procedures defined in the protocol (e.g. inclusion/exclusion criteria, timeliness of visits) and adverse event recording and reporting.

The Study Coordinator, or, where required, a nominated designee of the Sponsor, shall carry out a site systems audit at least once during the course of the study and an audit report shall be made to the TSC.

Study data

Monitoring of study data will include confirmation of informed consent, source data verification, data storage and data transfer procedures, local quality control checks and procedures, back-up and disaster recovery of any local databases and validation of data manipulation. The Study Manager or suitable deputy, or where required, a nominated designee of the Sponsor, shall carry out monitoring of study data as an ongoing activity.

Entries on CRFs will be verified by inspection against the source data. A sample of CRFs (10%) will be checked on a regular basis for verification of all entries made. In addition the subsequent capture of the data on the study database will be checked. Where corrections are required these will carry a full audit trail and justification.

Study data and evidence of monitoring and systems audits will be made available for inspection by the regulatory authority as required.

Record retention and archiving

In compliance with the International Conference on Harmonisation (ICH)/GCP guidelines, regulations and in accordance with the University of Nottingham Research Code of Conduct, the Principal Investigator will maintain all records and documents regarding the conduct of the study. These will be retained for at least 7 years or for longer if required. If the responsible investigator is no longer able to maintain the study records, a second person will be nominated to take over this responsibility.

The Study Master File and study documents held by the Principle Investigator on behalf of the Sponsor shall be finally archived at secure archive facilities at the University of Nottingham. This archive shall include all study databases and associated meta-data encryption codes.

Funding source

This study is funded by the NIHR.

Participant stipends and payments

Participants will not be paid to participate in this study.

Chief Investigator:

(Name) ________________________________________________

Signature: ______________________________________________

Date: __________________________________________________

Co-investigator:

(Name) ________________________________________________

Signature: ______________________________________________

Date: __________________________________________________

Study Statistician:

(Name) ________________________________________________

Signature: ______________________________________________

Date: __________________________________________________

Effect of maternal vaccination and acquired (vertical/passive) immunity in children

Influenza vaccination in pregnancy offers benefit to the mother by reducing the risk of infection and the resultant complications. It has also been established that immunisation in pregnancy with trivalent, unadjuvanted, seasonal influenza vaccine does provide vertical immunity to the child through the cord blood. 17–19,22,24 However, the clinical impact of the finding is less clear, with some studies indicating benefit and others not. 16–19 The same is not true of a monovalent, two-dose schedule, new variant influenza vaccine with or without adjuvant, where the scientific evidence for vertical transmission of passive immunity, although likely, has not yet been established.

Rationale for the proposed study

Given the current risk profile of the emergent pandemic virus, it could be assumed that pregnant women would readily choose to be vaccinated. However, perception and response to threats and assessment of risk do not necessarily align in terms of human behaviour. Research has suggested that people tend to overestimate the likelihood and impact of rare events and underestimate for more common situations. 27,28 The public response to the measles, mumps and rubella (MMR) vaccination scare and its possible link to autism and Crohn’s disease is an example. Despite substantial and sound evidence to the contrary, many parents chose to refuse MMR vaccination for their children on the basis of a theoretical association that has by most scientists and policy-makers been dismissed, therefore exposing them to the risk of serious disease from measles, mumps or rubella. Another example of a similar response is that to whooping cough vaccine in the 1970s. Whether the low take-up of seasonal influenza vaccine in pregnant women in America is due to similar anxiety is uncertain; however, policy-makers will be concerned to ensure maximum uptake of the H1N1 swine flu vaccination. Therefore, evidence to support the approach may help encourage women to come forward for immunisation. If data were available which revealed that vaccination of pregnant women appeared to confer meaningful protection against swine influenza to their babies after birth, this would enable messages to pregnant women of potential vaccinees to be shifted from ‘evidence that you are likely to benefit and no evidence that your baby will be harmed’ to ‘evidence that you are likely to benefit and further evidence that your baby will also benefit’.

The researchers propose to assess the immunity conferred to infants of mothers who have been vaccinated against H1N1 swine influenza by obtaining umbilical cord blood samples at delivery and submitting them for serological analysis, comparing with unvaccinated mothers in the same birth cohort.

Additionally, the study also plans to assess the efficacy of vaccination of the mother at prevention influenza/respiratory illness in the infant. This will be by following the child up for a period after birth and taking nasal swabs to look for influenza/other respiratory viruses and comparing the rate of infection in those of mothers who were vaccinated versus those who were not.

Purpose

To improve policy implementation by providing clarity on the degree of protection transferred from the vaccinated mother to infant and to enable clinicians to provide pregnant patients with accurate information with which they can make an informed decision over whether to accept immunisation or not, and to allow public health messages to be strengthened.

FIGURE 1.

Observational study to investigate vertically acquired passive immunity in babies of mothers vaccinated against H1N1v (swine influenza) during pregnancy.

Objectives

Primary objective To determine the proportion of babies who have acquired passive immunity to A/H1N1v (swine influenza) born to mothers who accept vaccination as part of the national vaccination programme whilst pregnant (during the second and/or third trimesters) against the novel A/H1N1 swine influenza virus (exposed group), compared with unvaccinated (unexposed) mothers.

Secondary objective To record and investigate influenza-like illness during winter 2009–10 and also to flag for long-term follow-up of babies born to mothers who take part in the study.

Study configuration

Observational cohort study across three centres [Queen’s Medical Centre and City Hospital, Nottingham, and Leicester Royal Infirmary (University Hospital Leicester)].

Full randomisation is not possible within this study, as the participants will have previously been vaccinated (exposed group) as part of the national swine influenza vaccination programme or not (in the case of the unexposed).

Recruitment will be at delivery. Although the researchers would prefer to recruit prior to delivery, they recognise that this has real practical difficulties, especially ensuring that those consented antenatally are sampled at delivery. Therefore, it is considered that recruitment will have to occur at parturition. For these reasons it is probable that the majority of participants will be women at term; however, those in the earlier stages of pregnancy (second or third trimester) and presenting in premature labour will be eligible for recruitment.

For the secondary objective (protective effectiveness of the vertical immunity provided to the baby from the maternal H1N1 swine vaccine against acquisition of A/H1N1 swine), the mother will be contacted after birth by the research midwife and provided with nasal swabs to obtain samples if the child becomes symptomatic with a influenza-like illness. The mother/parents will be provided with an information sheet including diagrams on how to do this and will also be trained by demonstration in taking these. With the participant’s consent, intermittent follow-up calls will be made to the mother over the period to 31 July 2010 to reinforce and remind her about swabbing if the child becomes ill.

With maternal consent a flag will be applied to the ONS record of the mother and child (from both the vaccinated and unvaccinated groups).

Statistical analysis will be undertaken in two stages as a minimum – firstly when all the cord samples (anticipated December 2009) have been obtained and secondly when the nasal swabbing sampling is complete (spring 2010).

Randomisation and blinding

As such, because the ‘exposed’ participants to be used in the study will be those who have already been vaccinated against H1N1 swine and the ‘unexposed’ participants controls will be those who have not, it is not possible to randomise at this primary selection level. However, it will be important to avoid the introduction of bias at this stage. It is possible that those accepting vaccination (exposed) as part of the national programme may represent a different group from those who do not/who have not yet been offered it (unexposed). For this reason, data at the point of recruitment on age, ethnicity, socioeconomic status, gravida, parity and pre-existing medical history will be sought to allow further analysis/enable potential confounders to be adjusted for. This should help to reduce the effect of any biases.

For obvious reasons the subjects cannot be blinded as they will know if they have been vaccinated or not and therefore likewise it will not be possible to blind the midwife recruiting/delivering the baby and taking the cord blood sample for the primary objective. Additionally for the secondary objective it will not be possible to blind the mother for nasal sampling. However, the cord blood serological analysis and nasal swab PCR determination will be blinded to the laboratory by use of unique code allocation to sampling. The results will then have to be unblinded at this point in order to allow statistical analysis. The Study Statistician will not have any direct contact will any of the participants.

Unique codes will be applied to the sample request forms and media with no other identifying data so that the laboratory will be blinded. The code will be linked to a separate participant study record that will be sealed and stored securely, and will be accessible to the study team only after receipt of the result in order to allow linkage and statistical analysis. (See maintenance of codes below for further detail.)

Enrolment will be coordinated by research midwives at the respective delivery units as previously listed above, with support from the usual care teams (midwife/obstetrician).

Unblinding

Unblinding of the cord sampling/nasal swab PCR results will occur once they are received to allow linkage to the participant study record, entry into the database and subsequent statistical analysis. The researchers will statistically analyse the results as the investigation proceeds to provide interim analysis.

Trial Steering Committee composition

The TSC will comprise the Chief (Principal) Investigator, the co-investigators and the Study Statistician (as listed earlier). Additionally, the study group will endeavour to engage a research consultant neonatologist and general practitioner (GP) to the TSC.

Trial Steering Committee terms of reference

1. To monitor and supervise the progress of the study towards its interim and overall objectives.

2. To review at regular intervals relevant information from other sources (e.g. related studies).

3. To consider the recommendations of the Ethics Committee.

4. In the light of 1, 2 and 3 above, to inform the NIHR Board and GSK on the progress of the investigation.

5. To advise the NIHR/GSK on publicity and the presentation of all aspects of the study.

End of the study

The end of the study will be date based and is to be 31 July 2010.

Recruitment

The study setting will be at the antenatal clinics/obstetric units of the three hospitals listed previously (Queen’s Medical Centre and City Hospital, Nottingham, and Leicester Royal Infirmary). These are all teaching hospitals. They have been chosen for the following reasons:

• large delivery numbers per year (in excess of 20,000 deliveries per year combined)

• established research bases for obstetrics and influenza/infectious diseases at each site

• close proximity to the researchers.

Clearly, this study is focusing on pregnant women and their babies, so all other groups will be excluded, i.e. men, older persons and older children. Pregnant women will be eligible to take part regardless of ethnicity or socioeconomic status.

Participants will be recruited from the delivery units of the above hospitals. The potential participant will be approached by a member of the patient’s usual care midwifery team for possible recruitment to the study. Information about the study will be on display in relevant clinical areas and advertised as widely as possible, so that pregnant women are aware of the research before recruitment. If the patient is interested in partaking then the usual care midwife in the relevant unit will then go through the details of the investigation with the potential participant of all aspects pertaining to participation in the study, including obtaining consent.

All of the maternity units have experience of recruiting women to studies during labour and this has been found to be an acceptable time to obtain consent. One of the key midwifery roles in labour is to act as the woman’s advocate. The usual care midwives are therefore very well qualified to decide when or if it is appropriate to approach a woman during her labour, depending on her level of distress and the possibility of clinical complications. The maternity unit will be paid for each cord sample taken but the money will not be paid directly to the usual care midwives – it will be used to improve general staff facilities in the unit.

The researchers have considered carefully whether pregnant women should be recruited antenatally, prior to parturition. However, logistically, it will be not possible to ensure that at delivery the samples of consented mothers are taken. For this reason, it is viewed that women will have to be enrolled at delivery.

Research midwives at each site will help to facilitate participation and coordinate the practical aspects of specimen collection.

If needed, the usual hospital interpreter and translator services will be available to assist with discussion of the study. The participant information sheets and consent forms will be available printed in other languages as far as is reasonably practical. It will be explained to the potential participant that entry into the study is entirely voluntary and that her treatment and care will not be affected by her decision. It will also be explained that she can withdraw at any time. In the event of her withdrawal it will be explained that the data collected so far will be retained.

Inclusion criteria

All pregnant women in the second and third trimester will be eligible to participate in the study if they present in labour; however, due to the short time scales required by the study commissioners to obtain results, the largest group recruited is likely to be those at term.

Women will be included regardless of age, social class, ethnicity, gravida and parity status, past and current medical history (including current medications), ethnicity mode of delivery and pregnancy outcome (live/stillbirth). However, all of these parameters will be noted for each participant to allow further analysis later.

The researchers have thought carefully about whether to exclude women who had underlying health conditions that might predispose them towards being vaccinated, as it is possible that their condition may alter the development of an appropriate immune response (e.g. if on immunosuppressants/renal impairment). However, the conclusion reached was that the information from this group was particularly important and therefore should be included, but to avoid the confounder effects adjustment at analysis may be required.

Exclusion criteria

Primary objective:

The main exclusion criterion is pregnant women in the first trimester/women delivering before the age of fetal viability (23 weeks and 6 days’ gestation).

Other exclusion criteria will be:

• incapacity to provide informed consent for participation refusal

• prisoners

• inability to take cord blood samples (e.g. cord blood needed for other clinical purpose, so none available for the study)

• involvement in another study entailing clinical interventions

• women who do not routinely live in the East Midlands.

Secondary objective:

• Inability to measure the outcome – this may be relevant if the woman is not normally resident in the UK or is moving abroad shortly after the child’s birth or is homeless and of no fixed abode. Compliance here may also be an issue. It may be that the possible participant may have such difficulty understanding the requirements of the secondary objective that it will not be practically possible to include her and her child.

• Refusal by the participant to agree to both parts of the study.

Expected duration of participant participation

Mothers up to 6 months in total, and their babies up to 5 months, depending on date of delivery (unless funding allows extension).

Removal of participants

Participants may have to be removed for the secondary objective if they cannot be contacted; however, this would only be accepted as a loss to follow-up if two reminder letters fail to re-engage the participant.

During the enrolment period, where pregnant mothers are being recruited for cord blood sampling at delivery and subsequent follow-up of the baby, if the participant withdraws consent, the researchers will seek to replace the lost individual if the number recruited to that date is insufficient to meet the power requirements of the study. After the enrolment period, if a mother subsequently declines to partake in nasal sampling then it will not be possible to replace that individual, as there will be no corresponding cord blood sample to provide comparison (exposed/unexposed to vaccine).

Abrupt termination from the study will not have safety implications to the participant.

Participants may be withdrawn from the study either at their own request or at the discretion of the Principal Investigator.

Informed consent

All participants will provide valid, written, informed consent. The Informed Consent Form will be signed and dated by the participant before they enter the study. The usual care midwife or team/research midwife will explain the details of the study and provide a Participant Information Sheet, ensuring that the participant has sufficient time to consider participating or not before obtaining consent if she wishes to partake. The midwife will answer any questions that the participant has concerning study participation. Consent will be obtained from the potential participant at the point of enrolment. Informed consent will be obtained from each participant before they undergo any interventions (including history-taking and cord blood sampling) related to the study. One copy of this will be kept by the participant, one will be kept by the Investigator (and stored with the participant’s study record), and a third will be retained in the patient’s hospital records.

Consent will be obtained through face-to face discussion with the potential participant.

The consent form will be kept with the case report form. Should there be a need later to amend the study (unlikely unless the study commissioner requests a subsequent modification) then each participant will be approached individually and reconsented by the research midwife or other member of the research team. This will be by written informed consent, which will again be stored in the same way as the original consent.

Explicit consent will be sought for both the primary and secondary objective participation, and the use and retention of the relevant data.

At the consent stage it will be made clear to the participant that the cord sample/nasal swab samples will, at the end of the study, be transferred to the University of Nottingham study bank, where they will be stored anonymously for use in other research, but that there will be no possible linkage to the individual participant. Participants will also be advised at this point that the sample will also not be available for subsequent use for clinical reasons, such as paternity determination or stem cell treatment of the baby if later required (as the sample collection and storage would not be appropriate to meet this need) and therefore if cord blood storage for this purpose was desired it would be for the participant to discuss and agree with the usual care team.

Compliance

Compliance with meeting the primary objective is unlikely to be a problem from a patient perspective. The research midwife coordinating on site will be based on the maternity unit as much as practicable in order to optimise enrolment and sample collection.

During the secondary objective phase (nasal swabbing), compliance will be promoted through the regular telephone contact of the participants and direct enquiry at that time whether the infant has had any respiratory infection in the intervening period and if so if they have been swabbed. Where there is a mismatch, the history of a respiratory infection without a swab having been taken will be noted and the mother reminded on the importance of obtaining a swab under these circumstances.

Criteria for terminating the study

The investigation may cease at any of the proposed centres if the adherence to the study protocol is unacceptable or the trust requests it. However, these are not anticipated to be likely and steps to correct problems would be identified first.

There are unlikely to be any specific safety issues with the cord blood sampling that would lead to complete study termination. (See below in the section on the transport and storage of the tissues re. the safe handling of the samples.) Likewise, this is also unlikely with the nasal swab sampling component, as there is established precedent from other studies for this method. Additionally, the mother/parents will be taught how to sample safely.

Possible external reasons for ceasing the whole investigation might include major and sustained business continuity problems at the relevant hospitals/investigating units, either as a direct consequence of the influenza pandemic or other issues. New information from another study source globally may render the need for the investigation unnecessary, unforeseen fund withdrawal and serology or PCR analysis problem may also lead to the investigation being terminated early. Additionally, if the vaccination programme were terminated this would prevent acquisition of vaccinated mothers and would necessitate ceasing the investigation. If, however, nationally, a safety issue with the vaccine were raised, it is likely that the study would need to be expanded – subject to additional funding availability.

Cord blood samples

Cord blood samples will be collected in appropriate sample containers. These will be subsequently collected by the research midwife on each site and packaged according to usual handling standards into appropriate containers designed for the safe transport of biological specimens (safe potential biohazard sample handling). Each sample and the accompanying request form will be labelled with the participant’s unique code only. As described elsewhere in this document, the unique code can be linked to the participant’s study record to allow linkage of the results but maintain anonymisation until then. Once packaged the samples will be transported (by post) to the Health Protection Agency (HPA) Centre for Infections, London, for analysis.

Nasal swab samples

Nasal dry swab samples will be collected in appropriate sample containers. These will be subsequently packaged into appropriate containers designed for the safe transport of biological specimens by the participant (having been previously shown how to do this). Each sample and the accompanying request form will be labelled with the participant’s unique code only. As described elsewhere in this document, the unique code can be linked to the participant’s study record to allow linkage of the results but maintain anonymisation until then. The samples will be transported (by post) to the HPA or Leicester Royal Infirmary, Clinical Microbiology Department, for suspension into viral transport medium and subsequent molecular analysis.

At the conclusion of the study, the cord blood and nasal swab samples will be transferred to the University of Nottingham Tissue Bank for anonymous storage for other researchers to use.

Cord blood serology

All cord blood serology samples will be analysed by MN and HI with the NIBRG121 virus [reverse genetics virus based on A/California/7/2009 (vH1N1) and A/Puerto Rico/8/34]. Samples collected at each study site will be centrifuged and separated into two aliquots. Samples will be tested in parallel.

The MN will be performed in 96-well format according to previously described protocols and standard operating procedures (SOPs) developed with the Respiratory Virus Unit (RVU), HPA Centre for Infections.

Elimination of complement (e.g. from fetal calf serum in culture medium) will be achieved by incubation of study sera and appropriate quality control sera (provided and chosen according to test virus by RVU: usually serum of ferret, sheep or human, with/without neutralisation activity) at + 56 °C/30 minutes. This step will be performed simultaneously for all study samples and control sera.

The MN analysis with the NIBRG121 virus will be performed as follows: a six-step twofold dilution series (covering titres 20–640) will be set up for each of the samples and control sera. After addition of a pretitred virus [usually around 100 × median tissue culture infectious dose (TCID₅₀) per well or 0.1-1 virus particle per cell] neutralisation will be performed by incubation of the virus/serum mixture at room temperature for 1 hour. After neutralisation, a suspension of Madin–Darby Canine Kidney (MDCK) cells will be added and the plates will be incubated for 16 hours at 37 °C in a CO₂ incubator. The remaining infectivity of virus after neutralisation is determined in an EIA format using a monoclonal antibody to detect expression of viral nucleoprotein. The amount of nucleoprotein expression is determined photometrically (optical density = OD₄₅₀) using a plate reader.

An OD reading for each dilution step for each sample will be used to calculate the titre. The titre will be reported as the reciprocal dilution at which 50% of the virus is neutralised (e.g. titre of 100). The MN analysis will be performed in duplicate (in separate runs on 2 days) for each sample. The two titres for each sample must not differ by more than a twofold serial dilution. In cases, where samples do not fall within this limit, a third analysis is performed and the two closest titres (which must be within a twofold serial dilution) will be reported.

The principle of the HI test is based on the ability of specific anti-influenza antibodies to inhibit haemagglutination of red blood cells (RBCs) by influenza virus haemagglutinin. The sera to be tested have to be previously treated to eliminate the non-specific inhibitors and the anti-species haemagglutination antigens. The experiment will be performed in accordance to protocols and SOPs established by RVU.

Elimination of non-specific inhibitors will be achieved by incubation of unknown serum samples and quality control sera (serum of ferret or human immunised with influenza virus) with neuraminidase [receptor-destroying enzyme (RDE) II: 18 hours/+36 °C followed by heat-inactivation 1 hour/+ 56 °C]. All samples – sera pre-vaccination and post vaccination and controls will be prepared simultaneously.

For the HI analysis with the NIBRG121 virus, samples and controls will be titrated in an eight-step twofold dilution series (covering titres 8–1024) and incubated with the HA antigen suspension (previously titrated to adjust the dilution at 4 haemagglutination units/25 µl, 50% end point). The HA antigen is not added to the well dedicated to the RDE quality control.

The mixture is incubated for 1 hour at room temperature and 25 µl of the 0.5% RBC suspension (turkey blood) are added. The reaction is left for half an hour at room temperature before reading.

The serum titre is equal to the highest reciprocal dilution, which induces a complete inhibition of haemagglutination. The titre of each quality control serum is close to the previously assigned value (within one serial twofold dilution limits). The RBC controls (RBC suspension without antigen) and the RDE controls do not produce any agglutination. Each serum sample is titrated in duplicate and individual titres will be reported (two for each sample). These must not differ by more than a twofold serial dilution. In cases, where samples do not fall within this limit, a third analysis is performed and the two closest titres (which must be within a twofold serial dilution) will be reported.

Nasal swab PCR

Nucleic acid extraction and real-time PCR (RT-PCR) will be performed by research staff at the Molecular Diagnostics Laboratory of the Leicester Royal Infirmary Microbiology Department, according to the UK National Standard Methods VSOP 25 (RT quadriplex PCR for the detection of influenza) and VSOP 29 (Swine-lineage Influenza A(H1)-specific fast RT-PCR). Equipment available for automated nucleic acid extraction includes the Corbett Xtractor Gene and the Qiagen Qiasymphony. RT-PCR analysis will be performed using the Corbett Rotor-Gene. The Leicester Microbiology Department is under the Pathology Directorate of the University Hospitals of Leicester/Leicester Royal Infirmary. Regular participation in performance evaluations, such as the National External Quality Assessment Service (NEQAS) and HPA, are SOP to maintain laboratory accreditation.

Methods

The overall programme manager, Dr Richard Puleston, Associate Professor of Health Protection at the University of Nottingham, will be responsible for the evaluation and analysis of the findings of the study. He will be assisted in this task by the statistical expertise provided by Dr Puja Myles and other statistical experts from the same unit. stata 11 will be the primary statistical analysis software, with additional use of Microsoft excel and other graphing/statistical software as appropriate (e.g. spss).

It is likely the researchers will use the following approaches.

Sample size and justification

The researchers have used a conservative power calculation based upon an estimated 20% seroconversion rate in unvaccinated women; in reality it may be closer to 10% as the cumulative clinical attack rate in the UK is still very low (probably < 2%) and so the true serological attack rate is probably < 4%. In addition underlying immunity in the age group 15–44 years (women of child-bearing age) is considered to be low. We have similarly estimated conservatively that seroconversion in vaccinated women will be 50%; in fact it is more likely to be 70% after two doses. Thus a power calculation based on 20% versus 50% is very conservative, and a more optimistic comparison would be 10% versus 70%.

Based on 20% versus 50%, with 80% statistical power, and 5% significance (two-tailed statistics), 38 subjects per group are required – total 76. However we anticipate that two-thirds of women will accept vaccine, so the ratio of unvaccinated–vaccinated subjects will be 0.5; allowing for this imbalance, a total study size of 89 subjects (59 vaccinated 30 unvaccinated) would be needed. Assuming a total of 89 subjects and more optimistic estimates of 10% versus 70%, the study would have 100% power to detect such a difference. To allow for possible losses during analysis or inadequate specimens, the researchers plan to recruit a maximum of 100 study subjects.

Assessment of efficacy

Primary efficacy end points will, as has previously been indicated, be based on the internationally recognised standard for immunity to influenza. Measures of seroconversion as specified by the EMEA–CHMP will be used. These criteria are formally accepted to be measures of seroconversion consistent with clinical protection against seasonal influenza in adults, and are used routinely for testing the immunogenicity of influenza vaccines in adults (HI titre ≥ 40 or single radial haemolysis > 25 mm²). MN tests may be used to assess responses to pandemic influenza but there are no recognised correlates of protection. Subjects achieving MN titres of ≥ 40 are may be considered as an immunogenicity end point. The extent to which these criteria are relevant to pandemic vaccines and to babies could both be debated but at present there are no widely accepted alternative measures, and these criteria are being used for the licensure of pandemic vaccines. They are therefore appropriate for this study.

Laboratory-confirmed influenza in symptomatic neonates will be measured by detection of H1N1 swine by validated PCR from clinical nasal samples.

The efficacy parameter will be difference in means for both the primary and secondary efficacy end points.

Procedures for missing, unused and spurious data

Missing data may fall into two categories: direct results from the primary and secondary objectives or indirect supporting information, such as date of birth or GP details.

Where the former is missing, every effort will be taken to track down what might be the problem, such as sample not actually taken through to result not being communicated. Where resolvable, the data will then be collated with the other findings. Where data items appear spurious, for example an implausible serological conversion level, clarification will be sought with the testing laboratory will be made to ascertain its veracity and, if necessary, request requantification, although the laboratory will have its own quality control procedures that should detect issues of this nature before reporting.

Where supporting data are missing the research midwives will endeavour to complete the data item either directly from the participant (e.g. number of children under 5 years in the household) or if more appropriate (e.g. details of the delivery) from the clinical record.

Where data items remain missing despite these efforts they will be treated as missing and will be noted as such in the analysis.

Definition of populations analysed

Interim analysis set, primary objective Analysis will be ongoing through the study. The relevance of the results as the study progresses will be based on the probability of obtaining such results for the number known.

Interim analysis set, secondary objective This will occur only if the national influenza activity on 31 January 2010 is above the normal baseline for the time of year.

Full analysis set, primary objective All individuals for whom a cord blood sample has been received at the close of recruitment and their subsequent delivery.

Full analysis set, secondary objective All infants for whom a nasal swab has been received by the termination date of the study, 31 July 2010, or the end of the pandemic, whichever is the sooner.

Efficacy will be assessed on the interim and full analysis set. Ineligible participants will be excluded before recruitment. However, if a participant becomes ineligible through withdrawal of consent then the research record for that person will be marked as such and noted at the time of analysis.

Reporting of adverse events

Participants will be asked to contact the study site immediately in the event of any actual or perceived SAE. All adverse events will be recorded and closely monitored until resolution, stabilisation, or until it has been shown that the study intervention is not the cause. The Principal (Chief) Investigator shall be informed immediately of any SAEs and shall determine seriousness and causality in conjunction with any treating medical practitioners.

Participant removal from the study due to adverse events

Any participant who experiences an adverse event may be withdrawn from the study at the discretion of the Principal Investigator.

Ethics committee and regulatory approvals

The study will not be initiated before the protocol, Informed Consent Forms and participant and GP information sheets have received approval/favourable opinion from the Research Ethics Committee (REC), and the respective NHS Research and Development (R&D) department. Should a protocol amendment be made that requires REC approval, the changes in the protocol will not be instituted until the amendment and revised Informed Consent Forms and participant and GP information sheets (if appropriate) have been reviewed and received approval/favourable opinion from the REC and R&D departments. A protocol amendment intended to eliminate an apparent immediate hazard to participants may be implemented immediately providing that R&D and REC are notified as soon as possible and an approval is requested. Minor protocol amendments only for logistical or administrative changes may be implemented immediately, and the REC will be informed.

The study will be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki, 1996, and the DH Research Governance Framework for Health and Social Care, 2005.

Informed consent and participant information

The process for obtaining participant informed consent or assent and parent/guardian informed consent will be in accordance with the REC guidance, and good clinical practice (GCP) and any other regulatory requirements that might be introduced. The investigator or their nominee (usual/research midwife or obstetrician) and the participant or other legally authorised representative shall both sign and date the Informed Consent Form before the person can participate in the study.

The participant will receive a copy of the signed and dated forms and the original will be retained in the Study Master File. A second copy will be filed in the participant’s medical notes and a signed and dated note made in the notes that informed consent was obtained for the investigation.

The decision regarding participation in the study is entirely voluntary. The investigator or their nominee shall emphasise to them that consent regarding study participation may be withdrawn at any time without penalty or affecting the quality or quantity of their future medical care, or loss of benefits to which the participant is otherwise entitled. No study-specific interventions will be done before informed consent has been obtained.

The Principal Investigator will inform the participant of any relevant information that becomes available during the course of the study, and will discuss with them, whether they wish to continue with the study. If applicable they will be asked to sign revised consent forms.

If the Informed Consent Form is amended during the study, the investigator shall follow all applicable regulatory requirements pertaining to approval of the amended Informed Consent Form by the REC and use of the amended form (including for ongoing participants).

Records

Data protection

All study staff and investigators will endeavour to protect the rights of the study’s participants to privacy and informed consent, and will adhere to the Data Protection Act, 1998. The CRF will only collect the minimum required information for the purposes of the investigation. CRFs will be held securely, in a locked room, or locked cupboard or cabinet. Access to the information will be limited to the study staff and investigators and relevant regulatory authorities (see above). Computer-held data including the study database will be held securely and password protected. All data will be stored on a secure dedicated server. Access will be restricted by user identifiers and passwords (encrypted using a one-way encryption method).

Information about the study in the participant’s medical records/hospital notes will be treated confidentially in the same way as all other confidential medical information.

Electronic data will be backed up every 24 hours to both local and remote media in encrypted format.

Insurance and indemnity

Insurance and indemnity for study participants and study staff is covered within the NHS indemnity arrangements for clinical negligence claims in the NHS, issued under cover of Health Service Guidelines (96)48. There are no special compensation arrangements, but study participants may have recourse through the NHS complaints procedures.

The University of Nottingham has taken out an insurance policy to provide indemnity in the event of a successful litigious claim for proven non-negligent harm.

Study conduct

The study conduct will be subject to systems audit of the Study Master File for inclusion of essential documents, permissions to conduct the study, Study Delegation Log, curricula vitae of investigative staff and training received, local document control procedures, consent procedures and recruitment logs, adherence to procedures defined in the protocol (e.g. inclusion/exclusion criteria, timeliness of visits) and adverse event recording and reporting.

The Study Coordinator or, where required, a nominated designee of the Sponsor, shall carry out a site systems audit at least once during the course of the study and an audit report shall be made to the TSC.

Study data

Monitoring of study data will include confirmation of informed consent, source data verification, data storage and data transfer procedures, local quality control checks and procedures, back-up and disaster recovery of any local databases, and validation of data manipulation. The Study Manager or suitable deputy, or where required, a nominated designee of the Sponsor, shall carry out monitoring of study data as an ongoing activity.

Entries on CRFs will be verified by inspection against the source data. A sample of CRFs (10%) will be checked on a regular basis for verification of all entries made. In addition, the subsequent capture of the data on the study database will be checked. Where corrections are required these will carry a full audit trail and justification.

Study data and evidence of monitoring and systems audits will be made available for inspection by the regulatory authority as required.

Record retention and archiving

In compliance with the International Conference on Harmonisation (ICH)/GCP guidelines, regulations and in accordance with the University of Nottingham Research Code of Conduct, the Principal Investigator will maintain all records and documents regarding the conduct of the study. These will be retained for at least 7 years or for longer if required. If the responsible investigator is no longer able to maintain the study records, a second person will be nominated to take over this responsibility.

The Study Master File and study documents held by the Principle Investigator on behalf of the Sponsor shall be finally archived at secure archive facilities at the University of Nottingham. This archive shall include all study databases and associated meta-data encryption codes.

Funding source

This study is funded by the NIHR. The secondary objective will also be supported by a grant from GSK subject to contractual agreements being completed.

Participant stipends and payments

Participants will not be paid to participate in this study,

Chief Investigator:

(Name) ________________________________________________

Signature: ______________________________________________

Date: __________________________________________________

Co-investigator:

(Name) ________________________________________________

Signature: ______________________________________________

Date: __________________________________________________

Study Statistician:

(Name) ________________________________________________

Signature: ______________________________________________

Date: __________________________________________________