Evaluation of Lactobacillus coryniformis CECT5711 strain as a coadjuvant in a vaccination process: a randomised clinical trial in healthy adults

Regarding white blood cells counts and differential, no differences were found between groups in each visit, nor within each group along the intervention (Additional file 1: Table S1).

Specific strains of probiotics interact with host cells and intestinal microbiota, and could thus have a role as immune modulators not only in patients with disease but also in healthy subjects under specific circumstances, such as an immune challenge as performed in this study. In fact, our findings showed that the consumption of Lc during two weeks before vaccination seems to be associated with an enhanced antibody response. However, the regular intake of this strain prior and following the challenge did not increase antibody titres but led to an increase in memory T helper lymphocytes.

L. coryniformis CECT5711 intake did not change the percentage and number of total T lymphocytes, including helper and cytotoxic T cells, B lymphocytes and NK cells. On the contrary, L. coryniformis CECT5711 in combination with L. gasseri CECT5714 consumed in a dose of 10⁶ cfu/g each during three months, led to an enhancement of NK cells in allergic children [24]. The effect of this combination on NK cells was also observed in healthy subjects after two weeks of treatment [25]. In agreement with the lack of effect on lymphocyte subsets in our results, several studies with different Lactobacillus strains supplementation in healthy individuals have also shown no significant effects on CD3+, CD4+, CD8+ and CD19+ percentages [25, 26]. Therefore, probiotics may affect the activity of certain immune cell types and not others, being the differences due to probiotic strain specificity.

There was an increase in memory T helper lymphocytes (CD3 + CD4 + CD45RO+) after the vaccine challenge in the group that consumed the probiotic strain during 6 weeks (PRO2), which might be linked to the establishment of immunological memory against the viral antigen. However, further research should be aimed to confirm the production of HAV specific memory CD4+ T cell clones, since specific memory cells can be reactivated after a secondary microbial exposure and are related to a long term protection [27]. The effect on lymphocyte subsets of a vaccination protocol against influenza virus while consuming L. fermentum CECT5716 (10¹⁰ cfu/d) was found to increase the percentage of helper and cytotoxic T cells after a two week challenge both in the placebo and treated groups [15]. The different timing in lymphocyte subset analysis between studies might explain why we did not observe the same increase in T cells, since we measured it after four weeks of vaccination. In addition, the nature of the vaccine antigen (bacterial or protein/ live or non live vaccines) and its administration could be main determinants in the immune response elicited after a vaccine shot [8] and thus relevant to evaluate differences between studies.

The specific production of antibodies in response to vaccination is considered as a useful measure which directly correlates with specific protection and the ‘gold-standard’ to determine the influence of probiotics on immunity [28]. In this regard, studies in animals and humans have shown the potential of probiotics to act as immune adjuvants [9], with an effect on specific vaccine antibody production in susceptible population groups such as children [29‐32] and elderly people [33, 34].

The current study is the first to use a vaccine challenge to assess the immune modulation exerted by the L. coryniformis CECT5711 strain in healthy adults. The effect of probiotics as vaccine adjuvants has previously been shown with other strains. The oral administration of the L. fermentum CECT5716 strain has been found to enhance the immune response of an anti-influenza vaccine and provide systemic protection from infection by increasing antigen specific IgA, but not IgG levels in 50 subjects [15]. In addition, the intake of B. animalis ssp. lactis and L. paracasei ssp. paracasei for 6 weeks have been shown to increase influenza vaccine-specific serum IgG measured 4 weeks after vaccination compared to placebo in 211 adults [10]. However, no effects in influenza A–specific IgG1 and IgG3 seroconversion measured three weeks after the vaccine were observed after the consumption of L. paracasei subsp. Paracasei 431 in 1066 healthy subjects. Protection rate after a seasonal influenza vaccine varies from year to year and these studies were performed in different campaigns, so the differences in the viral challenges between campaigns could also contribute to the different results found among these studies. Since rates up to 99% seroprotection were observed in Jespersen’s et al. study, the authors hypothesized that it might be difficult to observe a further increase in protection rates due to the probiotic intake [35]. In the current study, the consumption of the probiotic strain during 2 weeks in PRO1 induced an increase in Hepatitis A-specific antibodies after the vaccine, compared to the control group (P = 0.017). The immunological mechanism elicited after a vaccine challenge involves the activation of immature dendritic cells (DCs) by local inflammation, which take up the vaccine antigens and migrate to draining lymph nodes where the activation of T and B lymphocytes will take place. T cell help induces B cell differentiation into Ig secreting plasma cells that produce low-affinity IgG antibodies during this primary antibody response. Therefore, L. coryniformis CECT5711 might act as a coadjuvant of the antibody response in a vaccination protocol in healthy subjects when consumed before the vaccine challenge. This improvement in vaccine response could be relevant, since there is a low percentage of supposedly healthy individuals who exhibit an impaired response to the immune challenge of this vaccine and sometimes it needs an extra booster. In addition, EFSA states that “the stimulation of protective antibody titters in response to vaccination could be used to substantiate a health claim on the function of the immune system related to defence against pathogens” [11].

The fact that the increase of specific antibodies was not significant in PRO2 compared to placebo is difficult to explain. It might suggest that the probiotic consumption two weeks before vaccination works better as adjuvant of the humoral response than the continuation of L. coryniformis administration after vaccination; however, the level of specific HAV antibodies in PRO2 was at an intermediate level between the other two groups. In this sense, the continuous intake of this strain during 6 weeks in PRO2 could induce a higher T cell expansion, which is the main determinant of memory T cell responses and a weaker antibody response compared to the response induced when the strain was consumed only during 2 weeks in PRO1. In addition, regulatory T cell responses should be further evaluated in another study since an inverse relationship was observed between Tregs and antibody responses [36]. In fact, an enhancement of anti-cancer vaccine responses was observed in healthy adults following Tregs depletion [37]. No effects in inflammatory cytokines were seen when consuming the probiotic strain, in contrast to the placebo group, which showed higher levels of the pro-inflammatory TNF-α cytokine and the anti-inflammatory IL-10 four weeks after the vaccine, probably as an on-going reaction to the challenge [38]. In this context, the probiotic intake might have modulated the cytokine response to the vaccine. Although the early cytokine response after the shot was not evaluated, we could speculate that the probiotic intake might favour an early recovery of immunological homeostasis. On this basis, since mixed results were found depending on the timing and length of supplementation with the probiotic in relation to the viral challenge, we consider that one limitation of this study was the lack of certain additional times and immune measurements, such as innate immunity 2 weeks after vaccination and regulatory T cells at 4 weeks post-vaccination, in order to ascertain the role of the lactobacillus strain as an adjuvant for HAV vaccine.