Age-dependent systemic antibody responses and immunisation-associated changes in mice orally and nasally immunised with Lactococcus lactis expressing a malaria parasite protein

Gram positive food-grade bacteria such as lactococci have significant advantages over attenuated pathogens as vaccine delivery vehicles because of their inherently greater safety. Plasmodium falciparum merozoite surface antigen 2 (MSA2) was expressed in recombinant Lactococcus lactis both intracellularly and covalently anchored to the peptidoglycan of the cell wall (MSA2cP). Balb/c mice of different ages were immunised with the MSA2cP expressing L. lactis in a combined oral and nasal immunisation procedure. Serum IgG antibody responses to MSA2 were higher in young adult Balb/c mice compared to old mice and neonates. The elicited serum IgG antibodies reacted with native MSA2 on the surface of P. falciparum merozoites in an immunofluorescence assay. The serum IgG antibody isotypes in young adult mice were mainly IgG1, IgG2a and IgG2b, while IgG3 tended to be higher in old mice. IgA antibodies to MSA2 were also produced in young mice. Enlarged mesenteric lymph nodes, and more prominent lymphoid tissue in the lamina propria of the ileum and lymphoid follicles in the spleen, were observed in mice fed L. lactis. These findings are relevant for developing L. lactis as a vector to deliver vaccines in human populations.     

Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites

The 40-50 kDa merozoite surface antigen (MSA2) is a candidate molecule for use in a malaria vaccine. The gene for MSA2 from the 3D7 isolate of Plasmodium falciparum was amplified by polymerase chain reaction and cloned into the bacterial expression vector pGEX-3X to obtain a fusion protein of MSA2 with Schistosoma japonicum glutathione S-transferase. The recombinant fusion protein was used to immunize rabbits. After four injections, the sera had Western blotting and immunofluorescence titres of 10(-6). Immune sera, and immunoglobulin (Ig)G, F(ab)'2, F(ab) prepared from the immune sera, were assessed for their effects on the growth of 3D7 parasites in vitro by microscopy and a [3H]-hypoxanthine incorporation assay. The antibodies did not significantly inhibit red blood cell invasion and parasite growth when added to cultures as 10% v/v serum or as immunoglobulin preparations at concentrations up to 200 microg ml(-1). However, in the presence of IgG or F(ab)'2, but not F(ab), antibodies to MSA2, the proportions of red blood cells invaded by more than one merozoite increased significantly. Multiple invasion is attributed to merozoites cross-linked by bivalent antibodies, attaching to and subsequently invading the same red cell. These observations have a bearing on the evasion of host immune responses by the parasite and the use of full-length recombinant MSA2 protein in a malaria vaccine.     

Prevalence of attention deficit hyperactivity disorder (ADHD) in children presenting with self-inserted nasal and aural foreign bodies

Introduction

Attention deficit hyperactivity disorder (ADHD) is one of the commonest behaviour disorders in children, characterized by hyperactivity, inattention and impulsiveness. Tendency towards risk-taking behaviour and accident proneness is well recognized in these children. Accordingly, it could be hypothesized that children with ADHD are at increased risk for self-inserting foreign bodies, but a Medline search did not reveal any studies that investigated this possibility.

Objectives

To study the prevalence of ADHD among children seen with self-inserted foreign bodies.

Method

Children attending ENT service in a tertiary care children's hospital with self-inserted foreign bodies were assessed for the presence of ADHD. Two rating scales, Strengths and Difficulties Questionnaire (SDQ)-Parent Version, and Conners Parent Rating Scale (CPRS) were used to identify the cardinal features of ADHD.

Results

A total of 34 children, age 3-10 years, participated in the study. Majority (51.6%) were female. Nearly 25% had one or more previous incidents of foreign body insertion and 20% had previous accidental injuries that needed hospitalization. A prevalence of 14.3% for ADHD was identified, which is almost 3 times more than that reported in a previous study among children attending medical and surgical outpatient clinics (5.1%). High rates for abnormal hyperactivity scores were reported by parents, 37.4% with SDQ and 20% with CPRS. Although 64.7% of the sample was under 5 years, almost all children who were identified with ADHD belonged to 5-10 year age group, thus effectively excluding younger aged children who may have age related apparent hyperactivity.

Conclusions

Awareness of possible association between self-insertion of foreign bodies and ADHD is needed and an assessment for hyperactivity/ADHD is justified in such children, especially in those over the age of 5 years.     

Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations

A putative protective protein from Plasmodium falciparum merozoites, MSA2, was expressed in two different ways on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. The first display format exploits an LPXTG-type anchoring motif of the lactococcal proteinase PrtP to covalently anchor MSA2 to the genetically modified producer cells. In a second display format, MSA2 was fused to the peptidoglycan-binding domain (Protein Anchor) of the lactococcal cell wall hydrolase AcmA and was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells, termed Gram-positive enhancer matrix (GEM) particles. The L. lactis recombinants carrying covalently bound MSA2 were used to immunise rabbits through nasal and oral routes. The highest levels of IgG antibodies reacting with near-native MSA2 on merozoites was elicited by oral administration. Intestinal antibodies to MSA2 were produced only after oral immunisation. MSA2-specific T(h)-cell activation could be demonstrated. Based on these results, the immunogenicity in oral immunisations of MSA2, bound non-covalently to non-genetically modified L. lactis GEM particles, was compared with MSA2 that was bound covalently to genetically modified L. lactis. These two forms elicited similar titres of serum antibodies. The results illustrate the potential of using non-genetically modified L. lactis as a safe vaccine delivery vehicle to elicit systemic antibodies, thereby avoiding the dissemination of recombinant DNA into the environment.