Influenza remains a major cause of morbidity and mortality in the United States, resulting in approximately 19–36,000 deaths a year, and 200,000 excess hospitalizations (
Fiore et al., 2010). Influenza vaccination is the primary means of preventing influenza infection. Two types of vaccine are available - inactivated trivalent influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV). The Advisory Committee on Immunization Practices currently recommends annual universal vaccination of all children and adults greater than age 6 months (
Fiore et al., 2010). Although young children and the elderly suffer the greatest morbidity and mortality, influenza vaccination in healthy adults reduces both direct medical costs, such as physician visits and antibiotics uses, and indirect costs such as work absenteeism (
Nichol et al., 1999;
Wilde et al., 1999;
Fiore et al., 2010). The efficacy of influenza vaccine varies from year to year depending on the match between vaccine subtypes and circulating viral strains, patient’s age, and pre-existing immunity. In years in with a good match between vaccine and circulating virus, the efficacy of vaccine in healthy adults ranges between 80–90% for TIV (
Wilde et al., 1999;
Bridges et al., 2000;
Jefferson et al., 2007). Studies of LAIV in healthy adults have demonstrated a wide range of variability, with rates of clinical efficacy ranging between 40–80% (
Nichol et al., 1999;
Ohmit et al., 2006;
Monto et al., 2009). A number of alternatives to improve the efficacy of both LAIV and TIV have been explored in recent years including increasing to the dose of antibody in the vaccine (
Centers for Disease Control and Prevention, 2010), using alternative routes of administration such as intradermal injection (
Holland et al., 2008) and using immune adjuvants. Currently there is no FDA approved immune adjuvant for the influenza vaccine in the Untied States.
A number of studies in animals and humans have demonstrated the potential of probiotics to act as immune adjuvants. Lactobacillus species and other probiotics stimulate both the humoral and innate immune systems (
MacDonald et al., 2010). Healthy volunteers who received LGG prior to oral
Salmonella typhi vaccine developed higher IgA antibodies to the vaccine than those who received placebo (
Link-Amster et al., 1994;
Fang et al., 2000). Infants receiving LGG in conjunction with live oral rotavirus vaccine had higher rates of IgA seroconversion and higher numbers of rotavirus specific IgM secreting cells than infants receiving placebo (
Isolauri et al., 1995). Similarly, administration of LGG 1 week prior to an oral polio booster was associated with an increased poliovirus neutralizing antibody titers and poliovirus-specific IgA and IgG (
De Vrese et al., 2005). A recently published trial in newborns demonstrated that supplementation with a probiotic formula containing
Bifidobacterium longus and
Lactobacillus rhamnosus LPR was associated with a trend towards higher hepatitis B titers in infants immunized at birth (
Soh et al., 2010).
Studies in humans and animal support the role of Lactobacillus in the prevention of influenza infection. In a mouse model of influenza infection with an H1N1 strain, 3 days of intranasal exposure to LGG was significantly associated with a lower frequency of accumulated symptoms and a higher survival rate than control mice (
Harata et al., 2010). Using the same mouse model of influenza infection, oral administration of LGG or Lactobacillus TMC0356 for 19 days was associated with lower clinical symptom scores and pulmonary virus titers as compared to control mice (
Kawase et al., 2010). In studies of children in day care centers, administration of LGG was associated with a decrease in upper respiratory infections and length of infections (
Hatakka et al., 2001;
Hojsak et al., 2010). Probiotics have been investigated as immune adjuvants for TIV. In a placebo controlled trial using a supplement containing
Lactobacillus paracasei, elderly patients receiving the influenza and pneumococcal vaccines had an increase in the innate immune response and a decreased number of infections when compared with placebo (
Bunout et al., 2004). In another randomized placebo controlled trial, elderly patients who received a yogurt drink containing
Lactobacillus casei had higher influenza-specific antibody titers increased after vaccination (
Boge et al., 2009). In a trial of healthy adults,
Lactobacillus fermentum (CECT5716) improved influenza vaccine immunogenicity (
Olivares et al., 2007). Taken together these studies highlight the interaction of Lactobacillus and the respiratory mucosal immune system and the potential of LGG as an immune adjuvant with mucosally administered LAIV
Our study is the first randomized placebo controlled study to examine the effect of LGG on immune response to LAIV in normal healthy adults. The strengths of our study include the 93% completion rate, use of standardized endpoints and completion during a single influenza season. Although subjects receiving probiotics did not achieve greater seroprotection after administration of LAIV for the H1N1 and B strains, there was a significant improvement in those subjects receiving LGG for the H3N2 strain. The seroconversion achieved for any of the vaccine strains using LAIV was suboptimal and was particularly low (17%) for the H1N1 component that was new for the 2007–2008 season. Our suboptimal seroconversion rates are similar to those of DeVilliers
et al, who published the results of a large Phase III study of LAIV in the elderly (
DeVilliers et al., 2009). Overall seroconversion for all subjects receiving LAIV was 33.9% for the H3N2 strains and seroconversion rates and efficacy against infection with B strains were also low.
The study drug and LAIV were well tolerated in our study subjects. Although there were significantly more myalgias and decreased appetite in the placebo group, adverse events were similar to those observed in other LAIV studies (
Ohmit et al., 2009;
DeVilliers et al., 2009). One serious adverse event occurred in one placebo recipient who was hospitalized for a sinus infection on study day 58. Prior to this visit, the subject had not reported any sinus or upper respiratory symptoms.
Our study had several limitations. The sample size was small for this proof of concept study and we had insufficient power to detect small and moderate effects on vaccine responsiveness. Subjects previously vaccinated with TIV may have lower antibody responses to subsequent LAIV (
Sasaki et al., 2008) and this may have impacted the immune responses in the 49% of our subjects who had previously received TIV. We used traditional hemagglutinin inhibition assay to evaluate antibody titers. Several studies of pandemic H1N1 influenza in 2009 suggested that use of microneutralization assays or virus-free ELISA methods may be more accurate in measuring antibody responses (
Clark et al., 2009;
Alvarez et al., 2010).
Several important questions remain for future studies. LAIV is administered intra-nasally and it may be more relevant to measure the local mucosal response to evaluate the role of immune adjuvants targeting the mucosal surface. It may also be necessary to administer probiotics prior to LAIV rather than concurrently (
Boge et al., 2009). In addition, a more profound immune adjuvant effect may be demonstrated in groups which traditionally have a poor response to the influenza vaccine such as the elderly. The data generated from this study will be of critical importance to power future studies investigating the use of probiotics as immune adjuvants for mucosal vaccines.
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