Harold Marcotte

In situ delivery of passive immunity by lactobacilli producing single-chain antibodies.

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.

Lactobacilli Expressing Variable Domain of Llama Heavy-Chain Antibody Fragments (Lactobodies) Confer Protection against Rotavirus-Induced Diarrhea

BACKGROUND Rotavirus-induced diarrhea poses a worldwide medical problem in causing substantial morbidity and mortality among children in developing countries. We therefore developed a system for passive immunotherapy in which recombinant lactobacilli constitutively express neutralizing variable domain of llama heavy-chain (VHH) antibody fragments against rotavirus. METHODS VHH were expressed in Lactobacillus paracasei, in both secreted and cell surface-anchored forms. Electron microscopy was used to investigate the binding efficacy of VHH-expressing lactobacilli. To investigate the in vivo function of VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. RESULTS Efficient binding of the VHH antibody fragments to rotavirus was shown by enzyme-linked immunosorbent assay and scanning electron microscopy. VHH fragments expressed by lactobacilli conferred a significant reduction in infection in cell cultures. When administered orally, lactobacilli-producing surface-expressed VHH markedly shortened disease duration, severity, and viral load in a mouse model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried form. CONCLUSIONS Transformed lactobacilli may form the basis of a novel form of prophylactic treatment against rotavirus infections and other diarrheal diseases.

Characterization of oral lactobacilli as potential probiotics for oral health

INTRODUCTION Intestinal lactobacilli have been successfully used as probiotics to treat gastrointestinal disorders, but only limited data are available for the probiotic properties of oral lactobacilli to combat oral diseases. We aimed to characterize oral lactobacilli for their potential probiotic properties according to the international guidelines for the evaluation of probiotics, and to select potential probiotic strains for oral health. METHODS The study included 67 salivary and subgingival lactobacilli of 10 species, isolated from healthy humans. All strains were identified using amplified ribosomal DNA restriction analysis, tested for antimicrobial activity against oral pathogens, tolerance of low pH and bile content. Thereafter, the lysozyme tolerance and antibiotic susceptibility of 22 potential probiotic strains were assessed. RESULTS The majority of strains suppressed the growth of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus mutans, but none inhibited Candida albicans. The lowest pH tolerated by lactobacilli following 4 h of incubation was pH 2.5, but none of the strains grew at this pH. All strains tolerated a high concentration of lysozyme (10 mg/ml) and half of the strains tolerated a high concentration of human bile [5% volume/volume (V/V)]. Four Lactobacillus plantarum and two Lactobacillus oris strains expressed resistance to tetracycline and/or doxycycline. CONCLUSIONS Strains of L. plantarum, Lactobacillus paracasei, Lactobacillus salivarius, and Lactobacillus rhamnosus expressed both high antimicrobial activity and high tolerance of environmental stress. The absence of transferable antibiotic-resistance genes in L. plantarum strains remains to be confirmed. These results suggest a potential for oral lactobacilli to be used as probiotics for oral health.

Effective prophylaxis against rotavirus diarrhea using a combination of Lactobacillus rhamnosus GG and antibodies

BackgroundRotavirus is a worldwide cause of infectious infantile diarrhea that claims over 600,000 lives annually. Recently, two new vaccine candidates have been developed but their efficacy in developing countries, still remains to be proven. Oral delivery of specific immunoglobulins provides passive immunity and is a fast acting treatment for rotavirus diarrhea. Probiotic bacteria have also gained considerable attention lately as treatment for rotavirus diarrhea. Here we report an evaluation of the therapeutic potential of different probiotics and their combination with anti – rotavirus antibodies in a mouse model of rotavirus diarrhea.ResultsOf the six probiotic bacteria tested, Lactobacillus rhamnosus strain GG had the strongest influence in reducing prevalence, duration and severity of diarrhea and was therefore chosen for combination treatment with immunoglobulins. The combination treatment reduced the diarrhea outcome measures significantly, prevented histopathological changes and reduced the virus load in the intestines.ConclusionThe advantages associated with immunoglobulins and probiotics based therapy is that the treatment provides a rapid therapeutic effect and is cost efficient. These components do not require special storage conditions and could potentially complement the rehydration therapy that is currently used.

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

Integrative Expression System for Delivery of Antibody Fragments by Lactobacilli

ABSTRACT A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection.

Extended antimicrobial treatment of bacterial vaginosis combined with human lactobacilli to find the best treatment and minimize the risk of relapses

BackgroundThe primary objective of this study was to investigate if extended antibiotic treatment against bacterial vaginosis (BV) together with adjuvant lactobacilli treatment could cure BV and, furthermore, to investigate factors that could cause relapse.MethodsIn all, 63 consecutive women with bacterial vaginosis diagnosed by Amsel criteria were offered a much more aggressive treatment of BV than used in normal clinical practice with repeated antibiotic treatment with clindamycin and metronidazole together with vaginal gelatine capsules containing different strains of lactobacilli both newly characterised and a commercial one (109 freeze-dried bacteria per capsule). Oral clindamycin treatment was also given to the patients sexual partner.ResultsThe cure rate was 74.6% after 6 months. The patients were then followed as long as possible or until a relapse. The cure rate was 65.1% at 12 months and 55.6% after 24 months. There was no significant difference in cure rate depending on which Lactobacillus strains were given to the women or if the women were colonised by lactobacilli. The most striking factor was a new sex partner during the follow up period where the Odds Ratio of having a relapse was 9.3 (2.8-31.2) if the patients had a new sex partner during the observation period.ConclusionsThe study shows that aggressive treatment of the patient with antibiotics combined with specific Lactobacillus strain administration and partner treatment can provide long lasting cure. A striking result of our study is that change of partner is strongly associated with relapse of BV.Trial registrationClinicalTrials.gov: NCT01245322

Identification and characterisation of vaginal lactobacilli from South African women

BackgroundBacterial vaginosis (BV), which is highly prevalent in the African population, is one of the most common vaginal syndromes affecting women in their reproductive age placing them at increased risk for sexually transmitted diseases including infection by human immunodeficiency virus-1. The vaginal microbiota of a healthy woman is often dominated by the species belonging to the genus Lactobacillus namely L. crispatus, L. gasseri, L. jensenii and L. iners, which have been extensively studied in European populations, albeit less so in South African women. In this study, we have therefore identified the vaginal Lactobacillus species in a group of 40 African women from Soweto, a township on the outskirts of Johannesburg, South Africa.MethodsIdentification was done by cultivating the lactobacilli on Rogosa agar, de Man-Rogosa-Sharpe (MRS) and Blood agar plates with 5% horse blood followed by sequencing of the 16S ribosomal DNA. BV was diagnosed on the basis of Nugent scores. Since some of the previous studies have shown that the lack of vaginal hydrogen peroxide (H2O2) producing lactobacilli is associated with bacterial vaginosis, the Lactobacillus isolates were also characterised for their production of H2O2.ResultsCultivable Lactobacillus species were identified in 19 out of 21 women without BV, in three out of five women with intermediate microbiota and in eight out of 14 women with BV. We observed that L. crispatus, L. iners, L. jensenii, L. gasseri and L. vaginalis were the predominant species. The presence of L. crispatus was associated with normal vaginal microbiota (P = 0.024). High level of H2O2 producing lactobacilli were more often isolated from women with normal microbiota than from the women with BV, although not to a statistically significant degree (P = 0.064).ConclusionThe vaginal Lactobacillus species isolated from the cohort of South African women are similar to those identified in European populations. In accordance with the other published studies, L. crispatus is related to a normal vaginal microbiota. Hydrogen peroxide production was not significantly associated to the BV status which could be attributed to the limited number of samples or to other antimicrobial factors that might be involved.

Lactobacilli producing bispecific llama-derived anti-rotavirus proteins in vivo for rotavirus-induced diarrhea

AIMS Using genetically engineered lactobacilli, producing high avidity llama VHH domains (referred to as anti-rotavirus proteins; ARPs), to test the effect of multimeric antibody fragments as prophylaxis and therapy against rotavirus infection. METHODS Two ARPs, ARP1 and ARP3, shown to bind to different epitopes and act synergistically against rotavirus, were displayed on the surface of Lactobacillus paracasei as monovalent or bivalent proteins (mono- or bi-specific). RESULTS Although a nonsignificant difference was observed between lactobacilli producing bispecific ARP3-ARP1 and monomeric ARPs, lactobacilli producing bispecific ARP3-ARP1 were superior at reducing the rate of diarrhea when used for prophylactic and therapeutic intervention in a mouse model of rotavirus infection in comparison to nontreated animals. CONCLUSION Expression of bispecific antibodies in lactobacilli resulted in slight improvement of their efficacy. Furthermore, increasing the specificity would theoretically reduce the rate of appearance of viral escape mutants and would have a broader capacity to be effective against a range of viral serotypes.

Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries

Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.