A Randomized Trial of an Early Measles Vaccine at 4½ Months of Age in Guinea-Bissau: Sex-Differential Immunological Effects

Background After measles vaccine (MV), all-cause mortality is reduced more than can be explained by the prevention of measles, especially in females. Objective We aimed to study the biological mechanisms underlying the observed non-specific and sex-differential effects of MV on mortality. Methods Within a large randomised trial of MV at 4.5 months of age blood samples were obtained before and six weeks after randomisation to early MV or no early MV. We measured concentrations of cytokines and soluble receptors from plasma (interleukin-1 receptor agonist (IL-1Ra), IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, soluble urokinase-type plasminogen activator receptor), and secreted cytokines (interferon-γ, TNF-α, IL-5, IL-10, IL-13, IL-17) after in vitro challenge with innate agonists and recall antigens. We analysed the effect of MV in multiple imputation regression, overall and stratified by sex. The majority of the infants had previously been enrolled in a randomised trial of neonatal vitamin A. Post hoc we explored the potential effect modification by neonatal vitamin A. Results Overall, MV versus no MV was associated with higher plasma MCP-1 levels, but the effect was only significant among females. Additionally, MV was associated with increased plasma IL-1Ra. MV had significantly positive effects on plasma IL-1Ra and IL-8 levels in females, but not in males. These effects were strongest in vitamin A supplemented infants. Vitamin A shifted the effect of MV in a pro-inflammatory direction. Conclusions In this explorative study we found indications of sex-differential effects of MV on several of the plasma biomarkers investigated; in particular MV increased levels in females, most strongly in vitamin A recipients. The findings support that sex and micronutrient supplementation should be taken into account when analysing vaccine effects. Trial Registration clinicaltrials.gov number NCT 00168545


Non-specific effects of vaccines
The idea of vaccines having non-specific effects was first proposed in 1991 from a study in Senegal, West Africa. It was discovered that children receiving high-titre measles vaccine (HT) at 6 months of age had higher mortality than children who received the standard titre measles vaccine (STD) at 9 months of age 1 2 3 . The difference was found only for girls. A study from Haiti confirmed the effect 4 . Since children vaccinated with HT had lower mortality than their equivalents who had not received any measles vaccine, the difference in mortality between recipients of HT vaccine and STD vaccine was explained by a non-specific beneficial effect of the STD measles vaccine rather than a harmful effect of the HT vaccine 5 6 . The non-specific beneficial effect of STD measles vaccine on child mortality has been reconfirmed in many data-sets 7 8 9 10 11 .
Also the BCG vaccine is associated with striking effects on child mortality reducing mortality by about 50% 12 . Further, among BCG vaccinated children, having a BCG scar or a positive tuberculin reaction was associated with about 55% lower mortality in the following 12 months than among children who had a negative tuberculin reaction or who did not have a BCG scar 13 14 .
The effect of OPV is difficult to separate from the effects of BCG and DTP vaccines since OPV is normally given together with these vaccines. There have, though, been some periods without DTP in Bissau due to global shortage of vaccines, and we have compared the case fatality at the hospital for children who received only OPV and children who received both the prescribed OPV and DTP. Children having received OPV had 3-fold lower mortality than children having received both vaccines 15 . Data from an OPV vaccination campaign that took place in Guinea-Bissau also suggested a non-specific beneficial effect for the recipients 16 . Further, studies from Chile and the Soviet Union have suggested that OPV had a beneficial effect on mortality and morbidity 17 .
In contrast, DTP, HBV and inactivated polio vaccine (IPV) seem to exert a nonspecific detrimental effect on child mortality 12 18 19 20 21 22 23 , although the findings on DTP were considered controversial by a recent review 24 . Current studies indicate that the negative effect of DTP may be neutralized by a subsequent measles vaccination 25 26 . It is striking that all the vaccines with a non-specific beneficial effect are live, whereas the vaccines with an apparently harmful effect are killed. Results from animal studies have shown that attenuated live vaccines tend to induce a Th1 response and offer better protection against severe disease than the corresponding inactivated vaccines, which tend to induce a Th2 response 27 28 29 30 . So far, very few studies have examined whether these effects differ between male and female animals. One study reported that BCG-vaccinated female mice were better protected against malaria parasites than male mice 31 . There is therefore an urgent need to conduct studies that can help uncover the immunology behind the non-specific effects.

Sex-specific effects
All epidemiological studies carried out so far confirms the observation that non-specific effects are sex-specific 32 . Live vaccines (measles, BCG, OPV) have a beneficial effect that is particularly good for girls whereas inactivated vaccines (DTP, HBV, IPV) have a negative effect for girls. To date, there are no immunological studies which have examined whether routine vaccines affect the immune system differently for boys and girls.
We thus propose to study, in a randomised controlled trial of measles vaccination taking place in Guinea-Bissau, the immunology of non-specific effects of vaccination, and their interaction with sex. Specifically, among children who have received the 3 recommended doses of DTP, we will be able to compare the cytokine and antibody profiles of children who receive an early dose of measles vaccine at 4½ months of age with children who receive no additional vaccine at this age (please refer to methods).

Study site
The Bandim Health Project (BHP) has a demographic surveillance system in several districts of the capital of Guinea-Bissau, and the current population in these districts is around 80.000. All houses in the area are visited every month to register new pregnancies and births. The BHP visits all children at home every 3 months until the age of three years. Information is collected routinely on breastfeeding patterns, infections, hospitalisation, vaccination status, living with the mother, and ownership of pigs. The people of Bissau are travelling a lot due to extended families living in other parts of the country, cashew harvest, and trade. About 20% of the mothers are travelling at any given time. About 2.600 children are born in the BHP study area each year, and experience shows that we will be able to enrol about 80% of these in a measles vaccine trial i.e. about 2.000 infants per year.

Measles vaccination trial
We are currently conducting a randomised measles vaccination trial with the following schedule: The aims of the trial are to examine:  whether the negative effect of the DTP vaccine, which is administered by the EPI-programme at 6, 10 and 14 weeks of age, can be reverted by administration of a measles vaccine 4 weeks after the last DTP vaccination at around 4½ months of age  whether the standard-titre Edmonston-Zagreb (EZ) vaccine will be suitable for use in a very early two-dose schedule vaccinating at 4½ and 9 months of age, and  whether the standard titre Schwarz (SW) or standard-titre Edmonston-Zagreb (EZ) measles vaccine will be the best vaccine strain for use in a routine one-dose measles vaccination schedule and a two-dose measles vaccination schedule in terms of antibody response, protection against measles and child survival

Preliminary results, measles vaccination trial
The study was initiated in August 2003, and has currently recruited 2025 children. All routines are working well. A morbidity survey was implemented in August 2004.
It is too early to say anything about relative mortality in the three study arms. However, BHP has for many years registered all paediatric hospitalisations in Bissau which allows us to use hospitalisations as a morbidity outcome. Preliminary data on hospitalisations from the paediatric department in Bissau suggest that as predicted early measles vaccination protects strongly against hospitalisation for measles infection (see Table) but may also be beneficial, at least for girls, in protecting against hospitalisations for other causes. These preliminary results from a randomised trial corroborate our previous observational studies showing measles vaccine to be associated with a low female-male mortality ratio and DTP with a higher female-male ratio. The proposed study will be conducted as an immunological study performed among a subgroup of the participants in the above study.

Procedures, measles vaccination trial
Newborn infants are identified in the BHP registration system. To make sure that all children have received three doses of DTP before inclusion in the Two-dose trial, we contact the mothers of 6, 10 and 14 weeks old children, and remind them to go to the local health centre to receive the DTP vaccine. The formal inclusion in the Two-dose trial is at 4½ months of age. During the morning hours field-workers contact the mothers/guardians of the children to be included in the study. The field-workers explain about the study, perform a questionnaire, and obtain verbal consent (please refer to ethical considerations).
In the afternoon, the mothers/guardians of children present at the local health centre with the baby to receive oral (in Portuguese Creole) and written (in Portuguese) explanation about the study from the physician and to give consent for participation. The children belonging to arm I of the study will receive vaccination with the standard-titre EZ vaccine. At each visit to the health centre a physician will perform a medical examination. If the child is so ill that it needs to be hospitalised, it will only be able to participate in the study after it has recovered.

Additional procedures, proposed immunological study
In the proposed study it is planned to perform the first blood sample on the first contact at the health centre at 4½ months of age. At this age, all the children have received the third dose of DTP. On the same occasion, participants in arm I will receive a measles vaccine. Six weeks after this occasion (at 6 months of age), a blood sampled will be obtained at home. Children enrolled in the immunological study will be included in a morbidity study to be able to assess the possible correlation between differences in cytokine profile and morbidity.

Immunological analyses
The project provides a unique opportunity to perform detailed immunological analyses in a vaccination trial in a resource-poor developing country in a manner that will not be technically demanding nor taxing for the study population. Considering the limitation of only measuring a Th1 marker (IFN-g) in the face of growing evidence that a balance not only between Th1 and Th2 responses but also pro and anti-inflammatory networks are crucial for beneficial outcome of diseases and most probably vaccines, we will also measure IL-5, IL-13 as Th2, IL-10 as antiinflammatory and TNF-a and IL-1 as pro inflammatory markers. With the advent of the multiplexed particle-based flow cytometric assays for simultaneous measurements of cytokines or antibodies in small volumes of biological samples it is now possible to conduct immuno-epidemiological studies without compromising the population sample size 33 . This assay has been optimised for use in our setting, and is currently being used in Bissau in collaboration with Leiden University Medical Centre, The Netherlands. All blood samples will be collected by finger-prick in microtubes. Each participant to be blood sampled will have a blood smear prepared and examined for malaria parasitaemia. Participants with malaria will be treated according to local recommendations.

Whole blood culture
The blood will be diluted 1:10 with RPME-1640 medium and cultured in 96 well round bottomed plates. Stimulations will besides from control medium be performed with mitogens: PHA, anti-CD3+anti-CD28, and LPS. PHA is a strong stimulus for T cell derived cytokines particularly for IFN-g, IL-13 and IL-5. Anti CD3+CD28 is a strong stimulus for stimulating IL-10 production from T cells whereas LPS will provide us with information on responsiveness of the innate immune system mediated via Toll Like Receptor 4. Furthermore, the following antigens will be used: PPD and TT. Supernatants will be collected on days 1 and 3 and frozen at minus 80 degrees until use.

Cytokine determinations
The representative cytokines of the Th1, Th2 and regulatory T-cells will be measured simultaneously in the multiplexed particle-based flow cytometric assays. This newly developed assay system which in theory would allow the detection of more than 50 different cytokines in a small volume of 20 ul, is now operational at a level which allows the determination of now up to 8 cytokines simultaneously. The assay employs beads that are dyed with two fluorophores at varying ratios; different ratios therefore distinguish the test bead sets. Specific capture antibodies are coupled to specific bead sets and the different bead sets can be mixed together which means different cytokine capture antibodies can be added to the same sample. This refinement on the cytokine detection technology is of particular importance to immuno-epidemiological studies where volume of blood is a serious limiting factor.

Antibody determinations
Measles antibodies will be analysed at the Medical Research Laboratories in The Gambia by haemagglutination inhibition test (HAI), which has been in routine use for many years in The Gambia 34 35 .

Morbidity survey
The children enrolled in the immunological study will be followed with morbidity visits between 4½ and 9 months of age to assess disease episodes, consultations and vaccine adverse events. The morbidity survey collects information on diarrhoea, upper and lower respiratory tract infections, abnormalities of the skin, the general well-being of the child including measurement of temperature, consultations and hospitalisations. Within the first two weeks after vaccination the child will be visited 5 times, thereafter once per week. In case of diarrhoea, a faecal specimen will be collected 36 and analysed for parasites and rotavirus. In case of fever (an axillary temperature ≥37.5 o C), a blood smear will be collected for determination of malaria parasitaemia. In case of lower respiratory infection or other signs of severe disease, the mother will be urged to consult at the local health centre.
Further, the BHP monitors consultations at the local health centres and hospitalisations at the one paediatric department in Bissau. From these registers additional information on the children will be added.

HIV-infection in Bissau
The population in the study area has a high prevalence of HIV-2 infection (6-9%). However, very few children get HIV-2 infected and HIV-2 is therefore not expected to be an important confounder in the proposed studies. The prevalence of HIV-1 was 2.5% in the last survey before the war and is probably around 3-4 % at the moment among women of fertile age. Since the epidemic is recent, most mothers will be in the early phase of infection and vertical transmission is therefore expected to be somewhat lower than in other regions, presumably around 25%. We may ask for permission to carry out anonymous testing of blood samples that might be available from children who died to assess whether there is any indication of a differential effect for HIV-infected and uninfected children. Such permission has been granted in previous studies in Bissau.

Sample size Morbidity
With an expected diarrhea frequency of 6 episodes per year for children under 3 years (36), we can assume that each child will have at least one episode of diarrhea or fever between 4 ½ and 9 months of age. With 200 vaccinated and 200 unvaccinated children followed for morbidity, we will with a power of 80% be able to find a 20% reduction in infection incidence. Preliminary results indicate a 50% reduction in hospitalization rates for measles vaccination at 4 ½ months (table). Not only those who have taken the blood test, but all children (n = 5,755) included in the study will be registered in terms of hospitalizations. One third of the children enrolled at 4 ½ months will receive measles vaccine immediately, and 2/3 will receive measles vaccine at 9 months of age. With 765 vaccinated at 4 ½ months and 1,530 unvaccinated (who will receive the vaccine at 9 months) we will be able to find a 50% reduction in hospitalization with 80% power.

Immunology
Preliminary studies have shown a baseline level of IFN-g at 69 pg / ml. With 200 vaccinated and 200 controls we will be able to detect a 25% increase in IFN-g, and a 25% increase in IL-10 from 18 to 24 pg / ml.

Time table
The study is planned to begin in January 2005 with a recruitment period of about 8 months. Since season has a large influence on health in West Africa on both morbidity 37 and mortality 38 , as well as immunologic parameters such as delayed-type hypersensitivity, T-cells, thymus size and measles antibody levels 13 39 40 41 , it will be desirable to spread out the sample collection to cover both seasons of the year. Thus, we plan to include 25 children to be measles vaccinated per month, and 25 controls alternating from arm II and arm III.

Analysis
The analysis will emphasise changes in immunological parameters between the first and second sample for the two groups to assess the effect of a measles vaccine after DTP controlling for age related changes. Possible differences between boys and girls will be examined. This analysis is essentially an explorative investigation to detect immunological changes which might explain the differential effects of DTP and measles vaccine. At the same time, it will be investigated whether these potential differences in cytokine profile correlate with differences in morbidity between 4½ and 9 months of age when all children receive measles vaccine.

QUALIFICATIONS OF THE APPLICANT
The applicant has been employed as a medical researcher at the Statens Serum Institut, Denmark, and the Bandim Health Project, Guinea-Bissau, since 1994, and has completed a PhD thesis and several papers on specific and non-specific effects of measles and BCG vaccination 42 43 44 13 45 14 22 . Thus, the applicant has a sound basis for further research into these areas, and a great interest in continued research efforts that could lead to strengthening of the local Guinean research milieu, and hopefully leading to improved child health.

PUBLICATION OF FINDINGS
The analyses of data and the findings from the study will be discussed in the group of academics working on the project. The results will be made available to the Ministry of Health and health professionals in Guinea-Bissau, and we plan to publish the results in peer reviewed international journals.

ETHICAL CONSIDERATIONS
The two-dose study of measles vaccination was approved by the Ministry of Health in Guinea-Bissau, the MRC Ethical Committee and the Scientific Coordinating Committee at the MRC in The Gambia, and by the Danish Central Ethical Committee. The present protocol will be submitted to the same committees.
Mothers/guardians will be informed about the measles vaccination trial shortly before the child reaches 4½ months of age (see Procedures, measles vaccination trial). Since most mothers are illiterate there is no tradition for written consent in the study area. However, mothers who feel confident will be asked to sign, and those who cannot write will be asked to confirm their participation by fingerprint. Mothers accepting to participate will be asked to draw a lot to emphasise that the study is a randomised trial. The mothers will be asked to consent to participate after having received an explanation, which will contain the following information verbally (Portuguese Creole) and in print (Portuguese): Normally children receive one measles vaccine at 9 months of age. Since many children below 9 months of age risk to get measles we would like to find out whether it is best to get two doses of measles vaccine at 4½ and 9 months of age, or one dose at 9 months of age, or two doses at 9 and 18 months of age. We would like to test which of two types of measles vaccines will be the best for these schedules. Your child will either receive two doses at 4½ and 9 months of age, one dose at 9 months of age, or two doses of vaccine at 9 and 18 months of age. When your child is 4½ months of age you will draw a lottery, and if the child receives the first vaccination at 9 months of age, you will draw a lottery again at 18 months of age. Arm I: Your child has been selected by lottery to participate in the part of the study where your child will receive the first dose of vaccine at 4½ months of age, and the second dose at 9 months of age. We will take a blood sample by finger-prick from your child the first time he is vaccinated. Six weeks later when your child is 6 months of age, we will visit you at home to draw a second blood sample. We will take a third blood sample the second time your child is vaccinated at 9 months of age, and a fourth sample at 2 years of age to test whether he is protected against measles ("strength in the blood"). Arm II+III: Your child has been selected by lottery to participate in the part of the study where your child will receive the first vaccine at 9 months of age. At 18 months of age, you will draw a lot again to decide whether he should receive a second dose of measles vaccine or not. We will take a blood sample by finger-prick from your child at 4½ months of age at the first visit to the health centre. Six weeks later when your child is 6 months of age, we will visit you at home to draw a second blood sample. We will take a third blood sample the second time you visit the health centre at 18 months of age, and a fourth sample at 24 months of age to test whether he is protected against measles ("strength in the blood").
All arms: When we take blood samples, we will test whether your child has malaria, and treat him if necessary. At all visits to the health centre he will be clinically examined by a physician, and treated if necessary. You will not have to pay for this treatment. The results of these studies may contribute to better vaccination policies in the future. It is voluntary to participate, and you can withdraw from the study at any time. Do you have any questions about the study?