Vitamin A and Maternal-infant Flu Vaccine Response
Condition: Influenza
MeSH terms: Influenza, Human
Study type: Interventional

Summary: 
      Influenza viral infection can cause serious illness among young infants 0-6 months of age.
      However, inactivated influenza vaccine is not recommended for this age group but pregnant
      women can be vaccinated during 2nd - 3rd trimester to induce passive immunization of their
      infants. Nevertheless vitamin A deficiency is highly prevalent among pregnant women in
      Bangladesh, >50% pregnant women consume less vitamin A than the recommended level. Given the
      fact that both clinical and sub-clinical vitamin A deficiency impair vaccine specific
      immunity, in this proposed study, we aim to investigate whether maternal vitamin A
      supplementation improve influenza vaccine specific immune responses among pregnant women and
      the passive protection of their infants.

      In a placebo controlled clinical trial, sixty six mothers will be randomly assigned to
      receive either 10,000 IU vitamin A or placebo capsules weekly from second trimester to 6
      month postnatal period. At 26-28 weeks of gestation, all mothers will be vaccinated with
      inactivated, trivalent influenza virus vaccine. Maternal and cord blood will be collected for
      vitamin A and influenza virus specific IgG assessment. Colostrum and breast milk at 6-month
      will be collected for vitamin A and influenza virus specific secretory IgA assessment. Venous
      blood (2-3 ml) will be obtained from all infants at the age of 6 months for vitamin A and
      influenza virus specific IgG assessment as well as infants' nasal swab for influenza virus
      specific secretory IgA.
    

Detailed description: 
      Influenza morbidity affects the entire population and has an enormous impact upon the
      economic burden and the health care system, particularly causing serious illness among young
      infants 0-6 months of age. Recent studies show high rates of influenza illness and
      hospitalization among infants at this age group in industrialized countries (Chiu et al.,
      2002; Poehling et al., 2006) and influenza related childhood mortality is highest in young
      infants before 6 months of age (Bhat et al., 2005). Unfortunately, inactivated influenza
      vaccine is not recommended for this age group, but pregnant women can be vaccinated during
      2nd - 3rd trimester. Thus the strategy of passive immunization (from mother) of infants is
      the effective way to combat this infection among young infants. This approach adopted in US
      since 1997 and recommended by WHO since 2005.

      Upper respiratory tract is the portal of virus entry and primary site of replication (Gluck
      et al., 1994; Muszkat et al., 2000). Thus the importance of the presence of effective mucosal
      antibodies at the entry site of virus is essential to neutralize the virus and prevent
      infection (Meitin et al., 1994). Secretory IgA (sIgA) is the primary mucosal antibody,
      accounting for ~70% of the body's total antibody production (Brandtzaeg, 1994). sIgA has been
      shown to mediate nasal anti-influenza mucosal immunity (Renegar & Small, 1991) and therefore
      indicates the importance of vaccination strategies that trigger specific antibody production
      at mucosal surfaces as well as the importance of dietary nutrients to trigger the release of
      mucosal neuropeptides necessary for maintenance of the mucosal immune system (Renegar et al.,
      2001). A number of clinical trials examined the effect of vitamin A supplementation in humans
      on indicators of mucosal immunity. Supplementation to human immunodeficiency virus infected
      pregnant women is associated with improved gut permeability in their infants at 14 weeks as
      measured by lactulose/mannitol (L/M) urinary excretion test (Filteau et al., 1999). While
      vitamin A supplementation during pregnancy (Semba et al., 1999) or the early postpartum
      period (Filteau et al., 2001) show no effect on total sIgA in breast milk, rather vitamin A
      in milk has been shown to inhibit the growth of both enveloped and non-enveloped viruses
      (Clarke & May, 2000). However, total sIgA level of vitamin A-sufficient children has been
      detected significantly higher than that of vitamin A-deficient children (Lin et al., 2007).
      In animal model, vitamin A supplementation increases both total and antigen specific
      sIgA-containing cells in the mucosa, which in turn improves the survival rate (Nikawa et al.,
      2001) During early infancy breast milk is the predominant source of vitamin A and to date no
      study investigates the effect of prenatal vitamin A supplementation and vaccination on the
      vaccine specific mucosal sIgA responses in offspring. Although maternal IgA is not
      transplacentally transferred to the infants, but increased vitamin A and vaccine specific
      sIgA in breast milk as a result of maternal vitamin A supplementation may enhance infant's
      nasal sIgA responses that in turn may provide improved passive protection among young
      infants.

      In our recent study (Mother'SGift Project, funded by Thrasher Research Fund), we have
      detected that maternal influenza vaccination can reduce influenza illness substantially in
      young infants up to 6 months of age in Bangladesh (Zaman et al., 2008). On the other hand the
      prevalence of vitamin A deficiency among pregnant women in Bangladesh as well as in other
      developing countries is very high. More than half of the pregnant women in Bangladesh consume
      less vitamin A than the recommended dietary allowance and have low vitamin A status (serum
      retinol <1•05 μmol/L) with approximately one-fifth of them classified as deficient (serum
      retinol <0•70 μmol/L) (Ahmed et al., 2003; Lee et al., 2008). Nonetheless, there is
      conclusive scientific evidence suggesting several aspects of both innate and adaptive
      immunity are compromised by clinical and subclinical vitamin A deficiency (Stephensen, 2001).
      In another study, we observed a significant positive association between primary yellow fever
      viral vaccine specific lymphocyte blastogenesis / antibody responses and body vitamin A store
      (Ahmad et al., 2008). Thus in view of the potential immune regulatory roles of vitamin A, it
      is of interest to investigate whether maternal vitamin A supplementation along with influenza
      vaccination could be used as public health intervention strategy to combat both influenza
      infection and vitamin A deficiency simultaneously among mothers and young infants in
      developing countries.

      Research Design and Methods:

      Study Site: This study will be carried out at ICDDR,B; Dhaka, Bangladesh. Three urban
      maternity clinics located at Mirpur area of Dhaka city will be selected for approaching
      mothers who visit the clinic in the first trimester and who are the residence of the nearby
      community.

      Study Subject: The inclusion criterion is to enroll mothers at the beginning of second
      trimester (i.e. approximately 12 weeks of gestation), willing to stay in Dhaka during
      pregnancy and willing to admit in the clinic at delivery. The gestational age will be
      determined by self-reported LMP, which is likely be underestimated in most cases. A two-week
      variation will be acceptable in this proposed study. The exclusion criteria are: history of
      systemic disease, previous complicated pregnancies or of pre-term delivery, abortion,
      congenital anomaly, and hypersensitivity to influenza vaccine or receipt of the vaccine.

      Overview of Experimental Design: This is a placebo-controlled, double-masked and randomized
      trial. The overall study design is outlined in Figure 1, timeline and hypothetical duration
      of the proposed protocol related activities are illustrated in Table 1. To allow scheduling
      of the sample collection, storage and assay with the number of hired field attendants and lab
      facilities that will be available, we plan to enter approximately five pregnant mothers per
      week into this study in the first three-month period of the study (Table 1). Informed written
      consent will be obtained from the mothers. Sixty-six pregnant women will be randomized to
      receive either weekly 10,000 IU vitamin A or placebo (corn oil) capsules and blood will be
      collected for serum vitamin A assessment. Randomization will be stratified by three clinics
      using balanced block design. This weekly supplementation will continue until 6 months
      postpartum. The supplementing vitamin A dose to be used in this study is similar to the doses
      used weekly among pregnant women in other studies (Semba et al., 1999; Cox et al., 2006) and
      shown to be safe. Field workers will deliver the supplements to mothers, in person and will
      directly observe their consumption. At 26-28 weeks of gestation, all mothers will receive
      inactivated, trivalent influenza virus vaccine (GlaxoSmithKline; Dresden, Germany). Standard
      inactivated trivalent influenza A/H3N2, A/H1N1, and B strains as recommended by WHO for the
      Northern Hemisphere will be obtained from commercial sources for this project. Study
      physician will administer the vaccine intramuscularly. At delivery, cord blood and colostrum
      will be collected for Influenza specific IgG and sIgA assessment respectively as well as
      vitamin A concentration. Birth weight and length of the newborn babies will also be recorded.
      At 6-month, breast milk will be collected for vitamin A and influenza virus specific sIgA
      assessment and venous blood (2-3 ml) from infants (6 mo of age) will be obtained for vitamin
      A and influenza virus specific IgG assessment as well as infants' nasal washings for
      influenza virus specific sIgA assessment. Our proposed study will not hamper the existing
      prenatal care for the mothers such as folate/iron supplementation or existing infant
      vaccination program in Bangladesh.

      Randomization: Each mother will be given a unique randomly generated identification number,
      which will indicate the study group she is assigned to. The same ID will be labeled on the
      bottle containing 60 capsules. Field attendants will use these capsules for weekly
      supplementation ensuring the ID no of the mother and bottle ID are same. A third party (ACME
      laboratories, Dhaka Bangladesh) will prepare and supply vitamin A or placebo capsule in
      bottles, labeled with ID numbers from a list of numbers. The responsible personnel will keep
      it confidential until the end of study

      Sample collection: Peripheral blood from mother, cord blood, colostrum, breast milk,
      peripheral blood and nasal washings from infants will be collected as described in Figure 1.
      Nasal secretions will be obtained by introducing wicks (cotton applicators) into the nostril
      and gently rotating for 90s as described previously (Wright et al., 1996). The wicks will be
      extracted into 1mL PBS, centrifuged, and the supernatants will be collected. All samples will
      be frozen until tested simultaneously.

      Serological evaluation: We have chosen to investigate the humoral immune responses only to
      one influenza antigen because the purpose of this proposed study is to evaluate the effect of
      maternal vitamin A supplementation on the infants' passive antibody levels, thus it is
      prudent to investigate immune responses to one antigen. The following parameters of vitamin A
      and Influenza A specific humoral immunity will be tested.

      Vitamin A assessment: Vitamin A content in plasma, serum, colostrum and breast milk will be
      measured by standard HPLC method in place in the Nutritional Biochemistry Lab of LSD.

      Hemagglutination inhibition test [HAI]: HAI antibody titers will be measured by standard
      microtiter assay (Admon et al., 1997). 12 HA units of the selected Influenza A vaccine strain
      will be added to diluted pre-treated sera with receptor destroying enzyme (RDE, Sigma MO) and
      heat inactivation. Following incubation, washed chicken erythrocytes will be added until
      sedimentation ensues in the control wells. HAI titer is the inverse of highest dilution of
      serum exhibiting complete HAI.

      Influenza-specific IgG and sIgA ELISA: Influenza virus A specific IgG in serum/plasma and
      sIgA in colostrum, breast milk and infant nasal swab will be analyzed by ELISA as described
      previously with minor modification (Bruhl et al., 2000; Greenbaum et al., 2004). Briefly
      immunoplates will be coated with purified influenza antigen obtained from NIBSC at 2 ug/mL
      for overnight at 40C. and then diluted samples will be added for another overnight
      incubation. HRP-conjugated anti-human IgG (BD-Pharmingen, CA) or mouse anti-sIgA,
      peroxidase-labeled (Alpco Diagnostics, NH) will be used as conjugates. To construct standard
      curve, a serial dilution of pooled samples assigned with an arbitrary value will be
      introduced in each ELISA plate. Total sIgA in colostrum, breast milk and infant nasal swab
      will also be determined by EIA kit (Alpco Diagnostics) to adjust influenza A specific sIgA
      response with respect to overall sIgA secretion.
    

Intervention name(s): Vitamin A; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): 10000 IU Vitamin A or placebo per week; 10000 IU Vitamin A or placebo per week
Intervention MeSH terms: Vitamin A
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Prevention
Keywords: vitamin A; influenza vaccine; passive immunity; pregnant mothers; vitamin A suplementation; Influenza vaccination; Infant immune responses