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Immunogenicity and safety of different dosing schedules of trivalent inactivated influenza vaccine in pregnant women with HIV: a randomised controlled trial

Summary Background Standard-dose, seasonal, trivalent, inactivated influenza vaccine induces moderate-to-low haemagglutination-inhibition antibody responses in people living with HIV. This study assessed the immunogenicity and safety of different dosing schedules of inactivated influenza vaccine in pregnant women living with HIV in South Africa. Methods In this double-blind, randomised, controlled trial, we recruited pregnant women with HIV from seven antenatal clinics in Soweto, South Africa. Pregnant women were eligible if they were aged 18–38 years, infected with HIV, and had an estimated gestational age of 12–36 weeks. Women were randomly assigned (1:1:1), using a computer-generated randomisation list, to receive inactivated influenza vaccine containing 15 μg of each of the three seasonal influenza strains for that year, as a single dose, a double dose, or two single doses 1 month apart. Participants and study personnel were masked to group allocation. Haemagglutination-inhibition antibody responses were measured for all groups in the mothers at enrolment and at 1 month after each vaccine dose, and in the single-dose and double-dose groups within 7 days of birth in the neonates. Immunogenicity analyses only included women with visits 28–35 days apart and infants who were born at least 28 days after maternal immunisation. The primary was seroconversion rate to each of the vaccine strains in the mothers 1 month after completion of the dosing schedule, and the primary safety outcomes were frequency of local and systemic reactions. Safety was assessed in mothers and infants until 24 weeks post partum and analysed in all participants who received at least one dose of vaccine. This study is registered with ClinicalTrials.gov, NCT01527825, and is closed to accrual. Findings Between Feb 11, and June 6, 2013, 800 pregnant women living with HIV were enrolled and randomly assigned to the single-dose (n=266), double-dose (n=265), or two-single-doses (n=269) group. In the analysable population, seroconversion rates in mothers 1 month after the final vaccine dose were significantly higher in the double-dose group (n=230; ranging from 29% to 65% for the three vaccine strains) than in the single-dose group (n=230; ranging from 18% to 49%; p≤0·019 for the three vaccine strains), but were similar between the two-single-doses group (n=220; ranging from 23% to 52%) and the single-dose group (p≥0·20 for the three vaccine strains). Safety outcomes were similar in the three groups, except for more injection-site reactions in recipients in the double-dose group. Interpretation A regimen of double-dose inactivated influenza vaccine gave slightly greater immunogenicity than did a single-dose regimen in pregnant women living with HIV. However, immunogenicity in the double-dose group was still lower than historical data from the same setting in pregnant women without HIV. More immunogenic vaccines are needed for pregnant women living with HIV to enhance transplacental transfer of vaccine-induced protective antibodies to their newborn infants. Funding Bill & Melinda Gates Foundation.


Study inclusion criteria
Gestational age staging of the mother at enrolment was undertaken using a hierarchy of staging by fetal ultrasonagraphy (undertaken as part of standard of care), the last menstrual period of the mother and physical examination by palpation of fundal height.
(ii) Gestational age ≥12 weeks to <36 weeks documented by the approximate date of the last menstrual period and corroborated by physical/ sonargraphic exam. (iii) Documented to be HIV-infected on two assays prior to study-enrolment.
(iv) Able to understand and comply with planned study procedures.
(v) Provides written informed consent prior to initiation of study.

Study exclusion criteria
(i) Features of WHO clinical category 3 or 4 of AIDS at the time of enrolment.
(ii) Receipt of IIV, other than through the study, during the current influenza season documented by medical history or record. (iii) Receipt of any live licensed vaccine ≤28 days or inactivated licensed vaccine (except tetanus toxoid) ≤14 days prior to study-vaccine. (iv) Receipt of a non-licensed agent (vaccine, drug, biologic, device, blood product, or medication) ≤28 days prior to vaccination in this study or expects to receive another non-licensed agent before delivery unless study approval is obtained.
(v) Any significant (in the opinion of the site investigator) acute illness and/or oral temperature greater than or equal to 38C ≤24 hours prior to study entry.

(vi)
Use of anti-cancer systemic chemotherapy or radiation therapy ≤48 weeks of study enrolment or has immunosuppression as a result of an underlying illness or treatment.

(vii)
Long term use of glucocorticoids, including oral or parenteral prednisone ≥20 mg/day or equivalent for more than 2 consecutive weeks (or 2 weeks total) ≤12 weeks of study entry, or high-dose inhaled steroids (>800 mcg/day of beclomethasone dipropionate or equivalent) ≤12 weeks before study entry (nasal and topical steroids are allowed).

(viii)
Receipt of corticosteroids for preterm labour ≤14 days before study entry.
(ix) Receipt of immunoglobulin or other blood products (with exception of Rho D immune globulin) ≤12 weeks prior to enrolment in this study or is scheduled to receive immunoglobulin or other blood products (with the exception of Rho D immune globulin) during pregnancy or for the first 24 weeks after delivery.
(xii) History of a severe adverse reaction to previous IIV.
(xiii) Any condition that would, in the opinion of the site investigator, place the subject at an unacceptable risk of injury or render the subject unable to meet the requirements of the protocol.
(xiv) Pregnancy complications (in the current pregnancy) such as pre-term labour, hypertension (BP >140/90 in the presence of proteinuria or BP >150/100, with or without proteinuria or currently on antihypertensive medication) and pre-eclampsia. GMTs: geometric means titers; 95%CI: 95% confidence interval; HAI: hemagglutination inhibition. a significantly higher compared to baseline for all comparisons. b p-value calculated by pair student-t test.  26 23 Delivery related hospitalizations 54 (+2) 46 55 Hospitalization for infections 10 (+3) 6 7 Haematological related hospitalizations 1 0 1 Neurological related hospitalizations 1 0 0 Cardiovascular related hospitalizations 1 (+1) 1 4 Chronic condition related hospitalizations (+1) 1 4 Total number of maternal hospitalizations 91 (+3) 79 91 Total number of mothers hospitalized 83 (+9) 70 82 Only the primary diagnosis of each admission is included. a In parenthesis number of women randomised to the two single-doses group with an event before second vaccine dose was given or who missed second vaccination visit.   Only the primary diagnosis of each admission is included. a In parenthesis number of infants born to women randomised to the two single-doses group who were born before second vaccine dose was given.

Influenza in HIV-infected individuals
There is a paucity of data regarding the burden of influenza virus in developing countries, including those with a high prevalence of HIV. There is also limited data on Much of the differences in morbidity and mortality in HIV-infected individuals with influenza-associated pneumonia may be due to contribution of other co-existing pathogens. Although there are case reports of severe influenza illness in HIV-infected adults, in general the spectrum and severity of influenza associated illness have been described to be similar between HIV-infected and HIV-uninfected adults [13][14][15] . There is however a trend for HIV-infected adults being more likely to be hospitalized, evaluated in an emergency room or have an illness of ≥14 days than HIV-uninfected adults (21% vs. 0%; P=0.06) 15 . There also does not appear to be any evidence for clinical, immunological or virological progression of HIV during the course of an influenza season 16 .
In South Africa, the burden of influenza associated pneumonia has been characterized in HIV-infected children. The incidence of severe pneumonia in which influenza virus  18 . Furthermore, differences in the clinical spectrum of influenza-associated pneumonia include HIVinfected children remaining at risk of severe influenza pneumonia beyond early infancy, which is the major age-risk group in HIV-uninfected children, as well as there being more severe chest radiographic changes in HIV-infected children. The latter may be related to impairment of cell mediated immunity, which is key to controlling influenza virus replication and which is compromised in HIV-infected individuals.
Although there are no specific data on the burden of influenza illness in HIV-exposeduninfected infants, these infants are increasingly being recognized as having greater morbidity and mortality than infants born to HIV-uninfected mothers. The biological reason for this is yet to be fully characterized, although aberrations of the immune system of HIV-exposed-uninfected children have been described [19][20][21] . This may relate to exposure to HIV virions in-utero, which may affect responses to HIV-unrelated antigens.
Differences in immune responses to BCG vaccine in HIV-exposed-uninfected children were found by van Rie et al. 22 , however these findings were not confirmed in a more recent study 23  and HIV-exposed-uninfected infants 25 . This suggested that the heightened morbidity and mortality experienced by HIV-exposed-uninfected children was unlikely due to immunological aberrations. Another possibility for the increased morbidity and mortality, primarily experienced during early infancy in HIV-exposed-uninfected children may relate   Influenza vaccination of HIV-infected adults is also less likely to be associated with the generation of influenza specific memory B cells than in HIV-uninfected adults 35 .
It has been shown that two-doses of an inactivated Influenza A (H1N1) 2009 monovalent vaccine given 21-28 days apart to HIV-infected pregnant women was safe and immunogenic and that this regimen may achieve higher rates of antibody sero-protection than a standard single vaccine dose 36 .

Effectiveness of influenza vaccination in HIV-infected adults
In keeping with the modest immune responses observed to the sub-unit vaccines, a The incidence of "influenza illness", the outcome-measure which differed between studies in terms of diagnosis as well as in relation to severity, ranged between 21% in the Japanese study to 62% in the study by Fine et al. and in Italy 15,39,40 . The incidence of disease in the only placebo-controlled, randomized study by Tasker et al. 38 , which used an outcome of either respiratory symptoms with at least a four-fold increase in antibody titer and/or a positive viral culture, was 49% among unvaccinated and 29% in vaccinated individuals. This study too had limitations including relatively passive follow-up for influenza illness. Although impossible to make direct comparisons between these studies because of differences in case-ascertainment and outcome diagnosis, the high probably contributes to significant morbidity in HIV-infected adults.
The immunogenicity and efficacy of TIV in HIV-infected adults was only recently documented in an African setting. This placebo-controlled trial was approved by the University of the Witwatersrand Ethics Committee in 2008. This community-based randomized, placebo controlled trial reported that TIV was associated with a 75% reduction in influenza-confirmed illness 41 . The results of the study also confirmed the safety of TIV among African HIV-infected adults. The study, however, only included 7 women who were pregnant. In addition to no differences in solicited adverse event rates, there was also no difference in either CD4+ cell count changes or HIV viral load in those on antiretroviral treatment between TIV vaccinees compared to placebo recipients. This allayed previous concerns regarding the potential negative effect of TIV which centered around the observed transient increase in HIV viral load, even in HIV-infected individuals on HAART and who were virologically suppressed (viral load <400 copies/ml) 26,31 .

Decreases in CD4+ cell counts have also been observed in HIV-infected individuals
post-TIV vaccination 26 . These changes, however, even in past studies were infrequent (4-18%) and were considered to be clinically non-significant [27][28][29] . Preliminary data from 2011 Maternal influenza trials in HIV-infected and HIV-uninfected pregnant women supports the safety data described above: there were no significant differences in local or systemic adverse events reported by HIV-infected compared to HIV-uninfected pregnant women, and TIV did not have a significant impact on CD4+ cell and viral loads in HIV-infected participants.

Effect of pregnant-women TIV vaccination in mothers and young infants
The immunogenicity of TIV in pregnant women is similar to non-pregnant women, and documented a 36% reduction in clinical illness in vaccinated mothers 9 . This compared favorably to a meta-analysis on effectiveness of TIV in non-pregnant healthy adults in which a 67% reduction in influenza confirmed illness and 23% reduction of clinically diagnosed illness were documented 47 . There has, however, not been any study on the effectiveness of maternal immunization with TIV on influenza-associated morbidity and mortality either in the mothers or infants in African settings.
Despite the encouraging results on maternal immunization from Bangladesh, and the data supporting that TIV is efficacious mainly in HIV-infected non-pregnant adults, placental integrity and lower antibody levels in the fetus and newborn 48 . Furthermore, maternal hypergamma-globulinemia that is characteristic of HIV-infection may be associated with decreased neonatal antibody levels. This is supported by a study on the kinetics of measles antibody transfer during pregnancy in which although African women had higher total and measles-specific IgG levels, German women transferred a higher concentration of IgG to their infants 49 . This paradox is explained by the limited number of placental antibody receptors, resulting in IgG antibodies competing for available receptors and thereby decreasing vaccine-specific antibody transport 50 . In addition to the contribution of HIV, elevated IgG levels in African HIV-uninfected women may also be related to higher exposure to other pathogens as postulated in the above study 49 .
Finally, because preterm birth increase with HIV infection, chronic maternal disease or malnutrition, transfer of maternal antibodies which is gestational age dependent, may be more affected by maternal immunization in sub-Saharan Africa where these processes are common 50 . The need to corroborate the findings from studies in other population groups is supported by the potential differences in immunogenicity of vaccines. In addition, more importantly in the context of this proposal, are the potential differences which may exists between populations with regard to transplacental antibody transfer during pregnancy to confer "passive protection" during early infancy. Maternal

Study objectives
The randomized trial will evaluate the safety and immunogenicity of three different

Study population
General site description The study will be undertaken in Soweto (i.e. District D of Johannesburg, South Africa), an urban low-income community of 1.4 million Black-African inhabitants of a diversity of

Selection and enrollment of participants
HIV-infected pregnant women will be identified through the PMTCT program and enrolled at CHBH, where annually approximately 18 000 of the 28 500 pregnant women of Soweto will undergo at least one antenatal visit. The overall antenatal attendance rate for at least one visit, including that at primary health care clinics in the area is 99%. (iii) Documented to be HIV-infected on two assays prior to study-enrolment.
(iv) Able to understand and comply with planned study procedures.
(v) Provides written informed consent prior to initiation of study. (iv) Receipt of a non-licensed agent (vaccine, drug, biologic, device, blood product, or medication) ≤28 days prior to vaccination in this study, or expects to receive another non-licensed agent before delivery unless study approval is obtained.

Exclusion Criteria
(v) Any significant (in the opinion of the site investigator) acute illness and/or oral temperature greater than or equal to 38C ≤24 hours prior to study entry.
(vi) Use of anti-cancer systemic chemotherapy or radiation therapy ≤48 weeks of study enrollment, or has immunosuppression as a result of an underlying illness or treatment.
(vii) Long term use of glucocorticoids, including oral or parenteral prednisone ≥20 mg/day or equivalent for more than 2 consecutive weeks (or 2 weeks total) ≤12 weeks of study entry, or high-dose inhaled steroids (>800 mcg/day of beclomethasone dipropionate or equivalent) ≤12 weeks before study entry (nasal and topical steroids are allowed).
(viii) Receipt of corticosteroids for preterm labor ≤14 days before study entry.
(ix) Receipt of immunoglobulin or other blood products (with exception of Rho D immune globulin) ≤12 weeks prior to enrollment in this study or is scheduled to receive immunoglobulin or other blood products (with the exception of Rho D immune globulin) during pregnancy or for the first 24 weeks after delivery. (xi) Uncontrolled major psychiatric disorder.
(xii) History of a severe adverse reaction to previous TIV.
(xiii) Any condition that would, in the opinion of the site investigator, place the subject at an unacceptable risk of injury or render the subject unable to meet the requirements of the protocol.
(xiv) Pregnancy complications (in the current pregnancy) such as pre-term labor, hypertension (BP >140/90 in the presence of proteinuria or BP >150/100, with or without proteinuria or currently on antihypertensive medication) and pre-eclampsia.

Concomitant Medication Guidelines
Administration of any medication, therapies and vaccines will be documented in the study case report forms (CRFs). Concomitant medications will include all medications and vaccines taken during the following periods: 12 weeks prior to enrollment through end of study or early termination, whichever occurs first.
Receipt of any vaccines besides the study product will be collected throughout the study from enrollment to the off study visit. The administration of inactivated licensed vaccines should be at least 14 days prior to the administration of the study vaccine OR delayed until 14 days after the study vaccine has been administered. The administration of live vaccines is contra-indicated during pregnancy. After delivery live vaccines should not be administered ≤28 days of study vaccine administration. The use of these medications will be recorded throughout the study from enrolment to the off study visit.

Study description and assumptions
Blood samples will be undertaken as shown in the table of procedures.
Humoral immunity will be measured by hemagglutinin antibody inhibition (HAI) assay. In  Since TIV is consistently administered to HIV-infected individuals in the USA and somewhat consistently to pregnant women, we assume that some of the baseline HAI titers reflect prior immunization due to HIV or previous pregnancy, in addition to exposure to the wild type virus. In South Africa, TIV is infrequently used and, therefore, we anticipate that baseline HAI titers in South African HIV-infected and HIV-uninfected pregnant women will be lower than those observed in pregnant women in Colorado. In addition, all HIV-infected pregnant women received HAART during pregnancy in the USA, but not yet in South Africa. Since HAART is associated with improved immunogenicity of vaccines, we anticipate that the difference in HAI responses to TIV between South African HIV-infected and HIV-uninfected pregnant women will also be significant and perhaps higher than the difference in responses observed in Denver. We will also assess the effect of TIV administration during pregnancy on transplacental influenza-specific antibody transfer to the fetus. HAI titers, including determining the proportion with titers ≥1:40, will be measured in infants within one week of birth and at 8,  68 . Pregnancy has a tolerogenic effect on CMI and, therefore, we anticipate low baseline influenza-specific CMI followed by an increase after vaccine administration. The CMI evaluations in this study will provide novel information on influenza-specific CMI in pregnant women and newborn HIV-exposed infants.
In addition, we will compare the CD4+ cell counts before and after immunization. This study does not include a placebo arm, although, to maintain blinding, participants will receive at least one dose of a placebo vaccine. All participants will receive two vaccines at visit one, and one vaccine at visit 2 as follows: To offset the risk of developing severe influenza illness in study participants, the study will provide oseltamivir to all study participants known to be hospitalized for respiratory illness which is confirmed to be caused by influenza virus. Recommendations on the use of oseltamivir in pregnant women and young infants were recently published by the CDC and the study will use the same recommended doses 70 . It is anticipated that not more than 5% of all study participants will require treatment with oseltamivir.

Sample Size Considerations
The data from a parallel, albeit separate, study on the immunogenicity of TIV in a cohort of HIV-uninfected women and kinetics of transplacental antibody transfer to their infants will be used as a comparator for the immunogenicity and safety of single-dose TIV in HIV-infected women-infant dyads.
The sample size of HIV-infected women to be included in this safety and immunogenicity analysis has been powered at 80% and α<0.05 to detect at least a 30% difference in the Sample size calculation (1:1:1 ratio) based on 80% power and alpha less than 0.05. reasons. Blocks will be allocated consecutively to recruitment site when vaccine/ placebo are requested. Once a block of 30 numbers has been allocated to a recruitment site, the envelopes for allocated block with randomisation forms will be sent to recruitment site by pharmacist with the first set of vaccines prepared for the block. The randomisation forms, in envelopes with printed study number, will be pre-printed with the 4-digit study number and an alphabetical and colour code for vaccine/ placebo.

Estimated
Assignment to an arm of the study will be according to a random computer generated assignment by the study pharmacist. Study medication will be prepared by the pharmacist in syringes filled with either saline or with influenza vaccine as per randomization. Syringes for TIV and placebo administration will be identical to maintain double blinding.
Once an eligible participant has signed informed consent forms, the study doctor/ nurse at recruitment site will select the next randomization envelope (consecutive order), and select a pair of syringes with alphabetical code which corresponds to the pre-printed code on the randomization form. Vaccine/ placebo will be administered by a study-doctor or study-nurse in the deltoid muscle of each arm. Syringes containing TIV and placebo will look identical, however, for visit 1 vaccination; pharmacist will ensure that the syringes are labeled 'dominant' and 'non-dominant'. At visit 1, all participants will receive a dose of TIV in their non-dominant arm. Participants randomized to single dose and 2SD-TIV groups will receive placebo in dominant arm, and participants in double-dose arm will receive a dose of TIV in dominant arm.
At visit 2, all participants will receive an injection in their non-dominant arm: SD-TIV and DD-TIV participants will receive placebo, and 2SD-TIV participants will receive TIV.

Investigational product
In September 2012 WHO recommended a vaccine formulation containing the following influenza strains: TIV formulation: 0.5ml dose includes 15µg haemagglutinin (HA) of each of the 3 strains.

TIV
Using aseptic technique, 0.5 mL of TIV will be injected from a pre-filled syringe into a sterile 2ml syringe. A sterile needle for administration of TIV will be secured to the syringe. The study product will be labeled with one of five alphabetical letters assigned to TIV.

PLACEBO
Using aseptic technique, 0.5 mL of 0.9% NaCl solution will be drawn into a single-dose 2ml size syringe. The placebo will be labeled with one of the alphabetical letters assigned to placebo.
The study statistician, pharmacist and unblinded dispensing doctor will be the only members of investigative team who will be able to match alphabetical codes to TIV or placebo.

Study Product Acquisition and Distribution
TIV and 0.9% NaCl will be procured directly by RMPRU through the study grant from the local distributor of the vaccine. Purchase of TIV will be managed by the site pharmacist/dispenser or study coordinator. The study site pharmacists/dispenser will maintain complete record of all study products procured and dispensed. Sites will receive instructions regarding the final disposition of any remaining study products. Subject follow-up: The study will undertake active surveillance as detailed. The criteria for investigating for influenza illness will be as follows: Screening for possible influenza illness and other serious adverse events will include weekly home visits or telephonic contact of study participants from the time of enrolment of the mother. Home surveillance visits and or telephonic interviews will be undertaken by trained field workers, assisted by trained nurses, using structured questionnaires to interview for symptoms. The RMPRU has had previous experience in undertaking weekly home-visits as part of screening for illnesses during previous vaccine-interventional trials.
In addition, screening of hospital admission books to identify participants who have been admitted to hospital with a respiratory or other illness will be undertaken. Participants with influenza-like illness will have respiratory samples collected. Participants attending study center for unsolicited illness visits will be investigated for Influenza. In addition, participants will be sent weekly short message service (SMS) text as reminders of the symptoms of influenza illness with a request to attend the clinic if the mother or child fulfills pre-specified criteria suggestive of influenza illness.
Mothers will also be trained how to measure the child's axillary temperature and their oral temperature using temperature probes to be supplied by the study whenever suspecting fever.
Sample collection for influenza virus testing will involve nasopharyngeal aspirates (NPA) from infants and nasopharyngeal (NP) flocked swabs and oropharyngeal (OP) swabs from the mothers. The site has established standard operating procedures for these methods and has undertaken NPA in previous studies on respiratory viruses in children.
Although the yield between oropharyngeal swabs and NPA is similar for influenza virus in children, NPA has the advantage of obtaining samples which can be explored for other respiratory viruses as well.

Investigating for influenza virus infection
The proposal plans on having a low threshold for investigating for influenza illness, as the signs and symptoms may be very non-specific, especially in very young infants.
Additionally, the study will collect information of any symptom and signs that may be suggestive of influenza illness, to allow for analysis of influenza virus associated with a specific group of symptoms and signs. The final case definition of ILI and grading of influenza severity for infants and mothers will be done, a priori, during the development of the analysis plan. This will allow for consultation with other investigators from other countries where similar studies may be undertaken during the course of this study.

Criteria for investigating for influenza illness in infants
(i) Any documented fever (≥37.8°C on axillary measurements for <7 days duration) irrespective of presence of respiratory symptoms and signs; (ii) Fever (documented as ≥37.8C and/ or mother's perception that infant feverish/ hot) plus at least one sign/ symptom of acute respiratory infection Note: Detailed information of signs and symptoms will be collected on all children in whom samples are taken for influenza testing.

Criteria for investigating for influenza in mothers
(i) Any hospitalization for an acute respiratory illness of <7 days duration; (ii) Presence of fever (≥38 °C on oral measurements) or chills/ rigors or feeling feverish in past for <7 days duration OR; (iii) AND ONE of following for <7 days duration: a. cough or sore throat or pharyngitis, b. muscle aches or joint aches or headaches, e. chest pain while breathing or feeling short of breath or had difficulty breathing.
(iv) AND the absence of other diagnosis causing these symptoms.
(v) All participants attending clinic for unsolicited respiratory illness visits with at least one respiratory symptom/ fever documented in previous 7 days, irrespective of whether or not they fulfill ILI definition.
(vi) Alternatively, in the absence of fever and chills/rigors, the presence of symptoms from at least 2 of the remaining three groups of symptoms, in the absence of another diagnosis will require testing for influenza virus.
Samples for influenza virus testing should ideally have been obtained within 96 hours of symptom onset and no later than 7 days after initial onset of symptoms.

Influenza virus identification and molecular characterization
Viral particles will be detected in the NP/OP swabs and/or the NPA by a qualitative two step real time reverse transcriptase-polymerase chain reaction (rRTPCR) assay. Total nucleic acids will be extracted from 200µl of sample with a Biomerieux Nuclesin nucleic acid extraction robot (Biomerieux). Nucleic acids will be eluted in the standard elution buffer and stored at -70°C. Complementary DNA (cDNA) will be synthesized by using MultiScribe reverse transcriptase (RT) and random hexamers (both from Applied Biosystems). Reactions will be performed with 5µl of sample in a final volume of 20µL according to the manufacturers' recommendations. After incubation for 10 minutes at 25°C, RT is carried out for 30 minutes at 48°C, followed by RT inactivation for 5 minutes at 95°C. cDNA will be stored at -20°C. Primers and probe sets which target either the matrix gene or the hemagglutinin gene designed for the universal detection of type A and B influenza viruses, respectively, will be used in the PCR assays. Each sample will also be tested for a human positive control gene (Glyceraldehyde-3-phosphate dehydrogenase) to account for possible extraction and assay set up errors. All influenza A viruses will be further subtyped as either H1 or H3 using primers and probe sets that target the hemagglutinin genes of each virus. Testing for influenza virus will be undertaken at the RMPRU with quality assurance done at NICD. The choice of rRTPCR instead of viral culture for influenza virus is based on its increased sensitivity. rPCR typically detects an excess of 20% positive influenza specimens compared to viral culture. Both methods have equal specificity. The rPTPCR is performed under rigorous good laboratory practice conditions and the risk of contamination is minimal. The PCR targets are conserved areas of influenza A and influenza B genes and, therefore, we anticipate that antigenic drifts and shifts will not affect its diagnostic accuracy. Evaluation for genetic drift of influenza virus will be undertaken at NICD by sequencing of the HA1 subunit of the hemagglutinin and neuraminidase genes.
Samples obtained from infants and mothers will be archived at -70°C to examine for other respiratory viruses which may also contribute to respiratory illness. Funding for this aspect of the study will be sourced through alternate avenues. The specific methods used for influenza and other respiratory virus testing are included in a specific laboratory Standard Operating Procedure manual.

Breast-feeding practices
Breast-milk will be collected from breast-feeding mothers to assess the level of HAI IgA titers. Information on feeding practices will be collected at scheduled and weekly visits.

Immunogenicity studies
Humoral and cell-mediated immune (CMI) responses to influenza strains in the vaccine will be measured to assess the immunogenicity of TIV in pregnant women and their infants.
Hemagglutination inhibition assays will be performed on serum or plasma as described previously 71 . Briefly, specimens are treated with receptor-destroying enzyme (RDE) by diluting one part sample with three parts enzyme and incubating overnight in a 37°C water bath. The enzyme is inactivated by 30-min incubation at 56°C followed by addition of six parts 0.85% physiological saline for a final dilution of 1/10. HAI assays are performed in either V-bottom 96-well microtiter plates with 0.5% turkey erythrocytes, or in U-bottom 96-well microtiter plates with 0.5% guinea pig erythrocytes. Sera will be titrated against antigens from the influenza vaccine strains A H1N1 California, A H3N2 Perth and B Brisbane.
The importance of cytotoxic T lymphocytes (CTL) as a major contributor to defense mechanisms against influenza in humans has long been recognized [78]. In peripheral blood mononuclear cells (PBMC) from adults, influenza-specific memory CTL can be stimulated to rapidly develop effector function [79]. Granzyme B (GrzB) is a proapoptotic serine protease released from CTLs into influenza-infected target cells through a process facilitated by perforin, GrzB are transported across the cell membrane into the cytoplasm of the infected host cell. In animal models GrzB activity was reported to be the earliest and main contributor to CTL-mediated killing of influenza-infected cells [80].
GrzB is a key element in the enzymatic cascade that leads to apoptotic cell death and clearance of influenza virus from the lungs [80]. It has been shown that in older adults, GrzB activity in influenza-challenged PBMC correlates with protection against influenza and illness severity and that prior to influenza illness GrzB levels are lower in the influenza-stimulated PBMC from adults who subsequently develop influenza illness compared to those who do not [81,82]. Correlates of protection based on the cellmediated immune response may complement antibody responses in screening for the best vaccine candidates. GrzB activity will be measured in lysates of influenzastimulated PBMC. Brisbane); and with medium and phytohemagglutinin (PHA) (5 mg/ml) as controls. Spots will be visualized with a CTL ELISPOT plate reader. Background (non-specific) spots detected in the medium-containing wells will be subtracted from the wells stimulated with influenza antigens. Results will be reported as SFC/10 6 PBMCs.
The study will assess the effect of TIV administration during pregnancy on transplacental influenza-specific antibody transfer to the fetus. HAI titers will be measured in infants within one week of birth and at 8, 16 and 24 weeks of age. Using the 1:40 HAI titer as a defining threshold of protection against wild type influenza, we will determine the proportion of infants protected against influenza at birth and the duration of the protection conferred by transfer of maternal antibodies. We anticipate a higher proportion of protected infants among those born to vaccinated mothers compared with placebo recipients.

Processing of MATERNAL and INFANT samples
HAI assays. HAI titers against each strain in the vaccine will be measured on blood samples obtained from women at baseline, at 28-35 days post-first vaccination, 28-35 days post-second vaccine dose, at delivery (±1 week) and 24 weeks post-partum. HAI will be performed as described previously 71 . The antigens used in these assays will match the seasonal influenza vaccine serotypes obtained from the USA CDC. Detail SOPs for the assay are available from Dr. Weinberg's laboratory where assays will be undertaken. The kinetics of HA transferred to infants will be measure by examining HAI titers within the first week of life and subsequently at 8, 16 and 24 weeks of age. Nasopharyngeal Streptococcus pneumoniae colonization studies: NP specimen will be collected from the infants using Dacron swabs that will be placed in skim milk tryptoneglucose-glycerin (STGG) medium. Carriage will be measured by culture as well as by quantitative molecular analysis of pneumococcal density using real time PCR for the lytA gene and molecular analysis of serotypes present.
Nasopharyngeal viral carriage studies: Flocked NP and OP specimens will be collected from all infants at scheduled visits 4, 5 and 6 and placed in viral transport medium (VTM). Detection of different respiratory virus by PCR will be performed in these samples at a later stage.

Interaction between Streptococcus pneumoniae and influenza virus:
We will explore colonization by S. pneumoniae at the time of influenza-like illness in mothers and children to explore the idea that there may be a quantitative cutoff of S. pneumoniae colonization in the nasopharynx that is predictive of pneumococcal disease. Additionally, individuals with febrile illness will be investigated for S. pneumoniae co-infections by lytA PCR on serum or whole blood 73 . Although most infants in this study will receive PCV at 6 and 14 weeks of age, the overall effect of PCV immunization against pneumococcal

Toxicity Management
It is anticipated that vaccine-associated adverse events (AEs) will occur frequently, but that these will be minor local reactions and side effects. All AEs will be managed appropriately according to the situation.
Toxicities will be classified by the Division of AIDS ii. Local AEs, including pain, tenderness, redness, and swelling post vaccination.
iii. Systemic AEs, including feverishness, malaise, body aches (exclusive of the injection site), nausea, and headache post vaccination.
Iv. Adverse pregnancy outcomes, including maternal, fetal and infant complications.
ALL Grade ≥2 maternal adverse events regardless of association to vaccine will be recorded on the appropriate CRF.
Infant Grade 3 adverse events will be reported on the appropriate CRF. Management of the infant will be as per local standard of care.

Participant Management
Screening and study entry may occur on the same day; however, entry may be delayed up to 28 days after screening, if required. Mothers will remain in the clinic for at least 30 minutes after vaccination so that clinic personnel can observe for any potential adverse reactions to the vaccine. Equipment, supplies, and properly skilled medical personnel must be immediately available for emergency use in the event of an unexpected adverse reaction.
Report of vaccine-related local (redness, swelling, tenderness, itching) and systemic (fever, malaise, myalgia, nausea, headache, rash) adverse events will be solicited at day 7 and day 28. Serious Adverse Events (SAEs) will be reported as described below.
Antipyretics should not be routinely given in anticipation of adverse events after vaccination, but should not be withheld if symptoms occur. Antipyretics should be recorded on the memory aid and reported to the site staff. Breastfeeding is permitted in this study and the information recorded.
Infants will receive all the vaccine included the South African Expanded Program for Immunization as per standard of care. Infant AEs will be managed as per local standard of care. Infant Grade 3 AEs will be recorded at each study visit for the period of time elapsed since the previous visit.

Criteria for Study Discontinuation
The subject or legal guardian refuses further treatment and/or follow-up evaluations. The investigator determines that further participation would be detrimental to the subject's health or well-being.
The subject fails to comply with the study requirements so as to cause harm to him/herself or seriously interfere with the validity of the study results.

SAE Reporting Requirements for this Study
The SAE for which expedited reporting (within 24 hours of site awareness thereof) are required for this study will include: maternal death during pregnancy and after delivery.
During pregnancy, mothers will receive TIV or placebo in a blinded fashion. SAE will be reported without knowledge of treatment arm. At the end of the study, when the treatment groups will be known to the study team, SAE will be attributed to one of the active products or to placebo.
In addition to the SAE Reporting Category identified above, other AEs that must be reported in an expedited manner are: maternal hospitalizations other than for delivery, fetal loss, miscarriages, congenital anomalies, infant death through 24 or 28 weeks of life, whichever coincides with the end of the study follow up.

Grading Severity of Events
The Division of AIDS

Expedited AE Reporting Period
The expedited AE reporting period for this study is the entire study duration for an individual subject (from study enrollment until study completion or discontinuation of the subject from study participation for any reason).

Monitoring
Safety and tolerability of the study vaccine will be monitored by means of AEs and toxicity reports presenting laboratory and clinical data. All participants will complete a The data to be reviewed by the protocol team will be pooled across treatment arms. It is required that these data be entered into the database within 48 hours of the time at which the results of the laboratory tests or clinical examinations become available.
These reports will be discussed by the Core Team (comprised of the Study Chair and other co-investigators as well as the data manager and study-statistician) on monthly conference calls. Interpretation of vaccine-relationship to AEs will be based on the type In addition to monthly toxicity reviews by the Core Team, the study will be monitored by the DSMB. DSMB members will be independent of the study (except for the statistician) and have no perceived conflict of interest. The committee will meet annually either via conference call or face-to-face to review relevant data. The Chair of the DSMB will be mandated to report the DSMB's comments to the Gates foundation and Protocol Team.

Early stopping rules for safety
The following stopping rules will be applied in order to protect the study subjects from unnecessary exposure to the vaccine, should the safety profile prove unacceptable in this population. If at any time a subject experiences a life-threatening grade 4 toxicity, judged to be at least possibly related to the vaccine, the Core Team will request an independent review of the data by the DSMB.
If 20 or more subjects have experienced non-life-threatening grade 3 or 4 toxicities, judged to be at least possibly related to study treatment, then the study statistician will unblind the treatment assignments and summarize toxicities occurring in the subjects receiving placebo and those receiving TIV. In collaboration with an independent safety monitor who is a member of or appointed by the DSMB, the statistician will inform the study-team whether ≥30% of the subjects receiving either of the vaccine products have experienced non-life-threatening grade 3 or 4 toxicities which are at least possibly vaccine-related. If this has occurred, the study-team will request an independent review of the data by the DSMB. Otherwise, enrollment will continue.
Since unanticipated serious adverse reactions are not expected, enrollment will continue while the early safety review is taking place.
If the DSMB decides to halt the study for safety reasons and notifies the Study Team, the Study Team will notify the Gates Foundation, regulatory authorities and relevant stake holders immediately.

Human subjects protection: Institutional Review Board and Informed Consent
This protocol, the informed consent document, and any subsequent modifications must be reviewed and approved by the Human Research Ethics Committee (HREC) of the University of the Witwatersrand that is responsible for oversight of the study. Written informed consent must be obtained from the subject (mother). The informed consent will describe the purpose of the study, the procedures to be followed, and the risks and benefits of participation. A copy of the consent form will be given to the participant.
Should the consenting parent demise during the course of follow-up of the infant, the other parent of the child if available will be requested to consent for the continued participation of the child in the study. Should the mother have been the consenting parent and the father of the child be unavailable for consenting and not involved in the care of the child, the person taking on the role of primary care-giver to the child will be informed of the study. The continued participation of the infant will depend upon agreement thereof by the new primary care-giver, as indicated by signed consent, and in conjunction with notification thereof on a case-by-case basis to the HREC.

Subject Confidentiality
All laboratory specimens, evaluation forms, reports, and other records will be identified only by a coded number to maintain subject confidentiality. All records will be kept in a secured area. All computer entry and networking programs will be done with coded numbers only. Clinical information will not be released without written permission of the subject, except as necessary for monitoring by regulatory authorities or their designates.

Publication of Research Findings
Any presentation, abstract, or manuscript will be made available for review by the core study-team prior to submission. A detailed publication policy will be developed during the course of the study.

Biohazard Containment
As the transmission of HIV and other blood borne pathogens can occur through contact with contaminated needles, blood, and blood products, appropriate blood and secretion All infectious specimens will be sent using the ISS-1 SAF-T-PAK mandated by the International Air Transport Association Dangerous Goods Regulations-Packing Instruction 602. Refer to individual carrier guidelines (e.g., Federal Express or Airborne) for specific instructions.
Protection of staff: Because of the increased risk of influenza virus exposure by studystaff dealing with ill participants, special efforts will be undertaken to minimize the risk of influenza illness in staff. This will include training of staff in collecting illness-visit samples investigating for influenza, using standard operating procedures including the wearing of disposable gloves and the use of surgical masks. Additionally, all study-staff will be offered TIV vaccination and oseltamivir will be provided to any staff-member developing severe influenza illness during the course of the study. These measures will be further elaborated on in a SOP of managing staff with influenza-like illness.

Safety
For each group of mothers, the proportion of subjects who experience grade 3+ adverse events during the study will be presented and bounded by 90% confidence intervals.
This will provide 95% confidence that the true population proportion is no higher than the upper limit of the interval. The proportion experiencing grade 3+ adverse events judged to be treatment-related will also be shown. Since relatively few events of this severity are expected these analyses will be descriptive. However, should an unexpected epidemic of respiratory, CNS, febrile or exanthemous illness occurs during the study, there will more adverse events and a more formal analysis will be performed. In this case, the inclusion of the comparator group (single-dose TIV) will allow us to examine whether the symptoms associated with these adverse events are significantly more prevalent among participants receiving double or two-doses of TIV than among single dose TIV recipients and should attributed to the vaccination dosing schedule.

Immunology
In the mother: (i) Proportion of subjects with HAI titer ≥1:40 for each strain in the vaccine will be compared SD-TIV vs. DD-TIV and SD-TIV vs. 2SD-TIV and before and after vaccination.
(ii) Comparison of the proportion of subjects with ≥4-fold increase in HAI titer after vaccination compared to baseline for each strain in the vaccine for each study group.
(iii) Composite proportion of subjects with HAI titer ≥40 and 4-fold increase in HAI titer after vaccination for each strain in the vaccine per study group.
(iv) Comparison of the geometric mean HAI titers for each strain in the vaccine before and after immunization.
(v) Comparison of ELISPOT-measured SFC for each strain in the vaccine at baseline and after vaccination. (ii) Comparison of the geometric mean HAI titers for each strain in the vaccine.
(iii) Comparison of the maternal/fetal HAI titer ratio for each strain in the vaccine.
(iv) Proportion of infants with an ELISPOT result ≥50 SFC/10 6 PBMC in first week of life.

Primary immunogenicity analysis
Maternal: To compare the maternal TIV response rates at 28 days after each immunization to each of the three influenza vaccine strains (H3N2, H1N1, influenza B), between the study groups, the Chi-square test will be used. Logistic regression analysis may also be performed, should the need arise to control for certain covariates.

Infant:
Chi-squared tests will be used to compare the proportion of infants meeting the primary criterion for seroprotective levels of antibody to each of the three vaccine strains within 7 days of birth. Logistic regression analysis may also be performed, should the need arise to control for certain covariates.

Secondary immunogenicity analyses
Similar analyses to the primary analysis discussed above, comparing persistence of seroprotective levels of HA antibody to individual vaccine strains between infants of mothers in the three vaccine groups will be undertaken 8, 16 and 24 weeks.
In the mothers also similar analyses to the primary analysis will be done comparing the three TIV groups with respect to immunologic response at other time points will be performed.

Efficacy
The secondary analysis of efficacy of TIV will be analyzed as per intent-to-treat protocol.
This will involve all events occurring in mother and/or infant-pairs from the date of receipt of the study vaccine. A "per protocol" analysis on efficacy which will be limited to inclusion of HIV-infected mothers who received the correct randomized study-group within the study-defined window periods, the infant was born at ≥37 weeks of gestational age or birth weight of ≥2 500 g, and birth occurred at least 28 days after the mother had received study vaccine.
Efficacy will be measured using the following formula: VE(%)=(incidence rate in the single dose group-incidence rate in the double dose group)x100 incidence rate in the single dose group VE(%)=(incidence rate in the single dose group-incidence rate in the two doses group)x100 incidence rate in the the single dose group Only the first of any specified event being analyzed will be included in the analysis for any participant. Specific TIV efficacy endpoint outcomes will include: In the infant: (i) Laboratory-confirmed influenza illness in the infant from birth until 24 weeks chronological age.
(ii) Clinically diagnosed protocol defined influenza-like illness in the infant from birth until 24 weeks chronological age.
In the Mother: (i) Laboratory-confirmed influenza illness in the mother from birth until 24 weeks post-partum.
(ii) Clinically diagnosed protocol defined influenza-like illness in the mother from birth until 24 weeks post-partum.