Clinical and microbiological response of mice to intranasal inoculation with Lactococcus lactis expressing Group A Streptococcus antigens, to be used as an anti-streptococcal vaccine.

Protein subunit vaccines are often preferred because of their protective efficacy and safety. Lactic acid bacteria expressing heterologous antigens constitute a promising approach to vaccine development. However, their safety in terms of toxicity and bacterial clearance must be evaluated. Anti-Streptococcus pyogenes (S. pyogenes) vaccines face additional safety concerns because they may elicit autoimmune responses. The assessment of toxicity, clearance and autoimmunity of an anti-streptococcal vaccine based on Lactococcus lactis (L. lactis) expressing 10 different M protein fragments from S. pyogenes (L. lactis-Mx10) is here reported. Clearance of L. lactis from the oropharynges of immunocompetent mice and mice devoid of T/B lymphocytes mice was achieved without using antibiotics. The absence of autoimmune responses against human tissues was demonstrated with human brain, heart and kidney. Assessment of toxicity showed that leucocyte counts and selected serum biochemical factors were not affected in L. lactis-Mx10-immunized mice. In contrast, mice immunized with L. lactis wild type vector (L. lactis-WT) showed increased neutrophil and monocyte counts and altered histopathology of lymph nodes, lungs and nasal epithelium. Two days after immunization, L. lactis-Mx10-immunized and L. lactis-WT-immunized mice weighed significantly less than unimmunized mice. However, both groups of immunized mice recovered their body weights by Day 6. Our results demonstrate that L. lactis-WT, but not the vaccine L. lactis-Mx10, induces alterations in certain hematologic and histopathological variables. We consider these data a major contribution to data on L. lactis as a bacterial vector for vaccine delivery.

Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M-protein antigens derived from Group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N-terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N- terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N-terminal fragment of M protein, has been developed. Live bacterial-vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10-valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three- to 10-fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10-immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10-immunized mice did not differ significantly from that of unimmunized mice. Mx-10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis-based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

Importance of adhesins in the recurrence of pharyngeal infections caused by Streptococcus pyogenes.

PURPOSE: Pharyngo-amygdalitis is the most common infection caused by Streptococcus pyogenes (S. pyogenes). Reinfection with strains of different M types commonly occurs. However, a second infection with a strain of the same M type can still occur and is referred to as recurrence. We aimed to assess whether recurrence of S. pyogenes could be associated to erythromycin resistance, biofilm formation or surface adhesins like fibronectin-binding proteins and pilus proteins, both located in the fibronectin-binding, collagen-binding, T-antigen (FCT) region. METHODOLOGY: We analyed clinical isolates of S. pyogenes obtained from children with multiple positive cultures of throat swabs. We analysed potential associations between M types, clonal patterns, biofilm production and FCT types with their capacity of producing a recurrent infection. We genetically defined recurrence as an infection with the same M type (same strain) and reinfection as an infection with a different M type. RESULTS: No differences were observed between recurrent and reinfection isolates in relation to erythromycin resistance, presence and number of domains of prtF1 gene, and biofilm formation capacity; the only significant difference was the higher frequency of FCT-4 type among recurrent isolates. However, when all the factors that could contribute to recurrence (erythromycin resistance, biofilm production, presence of prtF1 gene and FCT-4 type) were analysed together, we observed that recurrent isolates have a higher number of factors than reinfection isolates. CONCLUSIONS: Recurrence seems not to be associated with biofilm formation. However, pili and fibronectin-binding proteins could be associated with recurrence because FCT-4 isolates which harbour two fibronectin-binding proteins are more frequent among recurrent isolates.

Association of vitamin D deficiency, season of the year, and latent tuberculosis infection among household contacts.

OBJECTIVES: Vitamin D (VD) enhances the immune response against Mycobacterium tuberculosis in vitro, and VD deficiency has been described in patients with active tuberculosis (TB). However, the role of hypovitaminosis D in the pathogenesis of early TB infection acquisition is unclear. We aimed to evaluate the association of VD deficiency, season of the year, and latent TB infection in household contacts (HHC), given that this is a potentially modifiable condition often related to nutritional deficiencies and lack of sun exposure. METHODS: We prospectively enrolled new pulmonary TB cases (n = 107) and their HHC (n = 144) over a 2-year period in Santiago, Chile. We compared plasma 25-hydroxycholecalciferol (25OHD) levels and examined the influence of season, ethnic background, living conditions, and country of origin. RESULTS: Over 77% of TB cases and 62.6% of HHC had VD deficiency (<20 ng/ml). Median 25OHD concentration was significantly lower in TB cases than in HHC (11.7 vs. 18.2 ng/ml, p<0.0001). Migrants HHC had lower 25OHD levels than non-migrants (14.6 vs. 19.0 ng/ml, p = 0.026), and a trend towards a higher burden of latent TB infection (52.9% vs. 35.2%, p = 0.066). Multivariate analysis found VD deficiency in HHC was strongly associated with being sampled in winter/spring (adOR 25.68, 95%CI 7.35-89.7), corresponding to the seasons with lowest solar radiation exposure. Spring enrollment-compared with other seasons-was the chief risk factor for latent TB infection in HHC (adOR 3.14, 95%CI 1.28-7.69). CONCLUSIONS: Hypovitaminosis D was highly prevalent in TB cases and also in HHC. A marked seasonality was found for both VD levels and latent TB in HHC, with winter being the season with lowest VD levels and spring the season with the highest risk of latent TB infection.