Patrick J. Lammie

MATERNAL FILARIAL INFECTION AS RISK FACTOR FOR INFECTION IN CHILDREN

Familial clustering of filarial infection was investigated through random house-to-house surveys of 643 individuals in Leogane, Haiti, an area with endemic Bancroftian filariasis. Children of infected mothers were 2.4 to 2.9 times more likely to be infected than were those of amicrofilaraemic mothers. Filarial-specific cellular responsiveness in amicrofilaraemic children born to infected mothers was lower than that in amicrofilaraemic children born to amicrofilaraemic mothers. No effect of paternal infection status was seen. The findings show that maternal infection is a risk factor for filarial infection in children and is associated with altered parasite-specific immune reactivity.

Cloning of the immunodominant 17-kDa antigen from Cryptosporidium parvum

Infection with Cryptosporidium parvum causes a self-limiting diarrheal illness in immunocompetent humans and is associated with the development of a serum IgG antibody response dominated by the 27-kDa and 17-kDa parasite surface antigens. Antibodies against the 27-kDa and 17-kDa antigens may serve as useful markers for past infection in population-based studies of the risk factors associated with Cryptosporidium infection. A recombinant form of the 17-kDa antigen would be useful both in epidemiologic studies and in studies of the role of the humoral response in immunity. We have partially purified and sequenced the immunodominant 17-kDa surface antigen from sporozoites, and we have cloned a 975 bp open reading frame from C. parvum that includes all of the 17-kDa antigen peptide sequences. We show immunologic identity between a recombinant form of the protein and the native 17-kDa antigen. We conclude that the carboxy-terminal fragment of the cloned protein is the authentic 17-kDa antigen.

The immunodominant 17-kDa antigen from Cryptosporidium parvum is glycosylphosphatidylinositol-anchored

Cryptosporidium parvum is a protozoan parasite of the intestinal epithelium that has caused numerous outbreaks of diarrheal illness in humans. During our studies of the host immune response to C. parvum infection, we noted that two of the immunodominant surface antigens of the sporozoite stage of the parasite readily extract into Triton X-114. We recently cloned the immunodominant 17-kDa surface antigen and suggested that the carboxy-terminal peptide sequence may satisfy the requirements for GPI anchor addition. In the work presented here, we were able to show that the 17-kDa antigen could be metabolically labeled in vitro with tritiated ethanolamine and that the antigen contained myo-inositol. The antigen was cleaved by GPI-PLD but not by PI-PLC and it could be converted to a water soluble form by chemical deglycosylation. We suggest that the 17-kDa antigen is indeed GPI anchored and that the anchor contains an acylated inositol and either a lyso-acyl- or a diacyl-glycerol. We are currently working to determine what role the anchor may play in the human immune response to this antigen.

Long-term suppression of microfilaraemia following ivermectin treatment.

Lymphatic filariasis has been difficult to control until recently because of the lack of a suitable drug for treatment. Ivermectin has proven safe and effective at reducing levels of circulating microfilariae. However, the apparent need to administer the drug every 6 to 9 months to keep microfilaraemia levels sufficiently suppressed to reduce transmission has been a major drawback to using ivermectin in community-based intervention programmes. In a study conducted in Haiti, we have found that high doses of ivermectin suppress microfilaraemia levels for 2 years. Our findings suggest that a single dose of ivermectin can reduce transmission of lymphatic filariasis for extended periods of time, thus eliminating the need for costly biannual treatment.

Acquisition and expression of humoral reactivity to antigens of infective stages of filarial larvae

Measurement of anti‐larval responses in filaria‐exposed populations may shed light on the natural history of exposure to Wuchereria bancrofti. Using serum samples obtained by a cross‐sectional survey of 172 individuals from two neighbourhoods in Leogane, Haiti, antibody responses directed against infective stage filarial larvae (L3) were assayed by enzyme‐linked immunosorbent assay (ELISA), immunofluorescence (IFA), and immunoblot for the presence of anti‐larval antibodies. ELISA results indicated that virtually all members of both neighbourhoods mounted an anti‐larval antibody response within the first five years of life, suggesting that exposure to infection is universal. In a multiple linear regression analysis that modelled antibody levels as a function of age, gender, microfilaria status, and neighbourhood (as a proxy for transmission intensity), isotype‐specific antibody levels were found to be significantly influenced by both age and neighbourhood. Antibodies directed against the surface of L3 also were age‐dependent; the prevalence of IgG antibodies detected by IF A was significantly higher in children than in adults. The prevalence of antibody recognition of 16–7 and 72–3kDa L3 antigens on immunoblots was significantly greater for serum samples from microfilaraemic than amicrofilaraemic persons. These results suggest that antibody responses to larval antigens are influenced to varying degrees by age, transmission intensity, and microfilaremia status.

Investigation of the influence of maternal infection with Wuchereria bancrofti on the humoral and cellular responses of neonates to filarial antigens

Epidemiological data indicate that maternal filarial infection might be associated with increased susceptibility to filarial infection in offspring. To examine the influence of maternal infection on development of antifilarial immunity in neonates, paired cord and maternal sera and mononuclear cells were collected in an area where Wuchereria bancrofti infection is endemic. Anti-filarial humoral responses (IgG, IgM and IgE) non-parasite-specific humoral responses (total IgE), proliferation induced by filarial antigen and production of cytokines (interleukin-2, interleukin-4 and interferon-gamma) were all monitored. Few cord serum samples had detectable antifilarial IgM of IgE and neither these responses nor total IgE levels differed as a function of maternal infection status. Of cord-blood mononuclear cells assayed, a relatively small proportion exhibited reactivity to filarial antigens. Based on these limited responses to filarial antigens, few neonates display evidence of in-utero sensitization to filarial antigens.

Immunologic characterization of jird lymphocyte responsiveness to Brugia pahangi ribosomal protein S13

Ribosomal protein S13 of the nematode Brugia pahangi is recognized by B and T cells from parasite-infected animals. To identify helper T cell sites on the protein, 15 overlapping synthetic peptides spanning the entire molecule (Bp17.4) were tested for their ability to stimulate lymph node and spleen cells of peptide-immunized and recombinant antigen-immunized jirds. Lymph node cells from animals immunized with peptides 6, 8, 9, 13, and 14, corresponding to Bp17.4 amino acids (AA) 50-70, 70-90, 80-100, 120-140, and 130-150, respectively, showed strong and specific responses to the homologous peptide, while only those lymph node cells from jirds immunized with peptides 8, 9, 13, and 14 proliferated in response to Bp17.4. These results suggest the existence of at least two T cell epitopes. Lymph node cells from infected jirds also responded to these peptides and to Bp17.4 (80,000 cpm). In contrast to the lymph node cells, spleen cells from microfilaria-positive animals failed to mount significant responses to any of the peptides or to Bp17.4. Splenic T cell responsiveness was restored upon removal of nylon wool adherent cells, suggesting active regulation of Bp17.4 reactivity. In liquid-phase competitive inhibition immunoassays, peptides 1 (AA 1-30) and 6 (50-70) blocked antibody binding and, therefore, these regions contain conformational antibody-binding sites. This model system should prove useful for analyzing regulation of epitope-specific responses in experimental filariasis.