Development of Onchocerca volvulus in humanized NSG mice and detection of parasite biomarkers in urine and serum.

BACKGROUND: The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice. CONCLUSIONS/SIGNIFICANCE: The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus

The Immunomodulatory Role of Adjuvants in Vaccines Formulated with the Recombinant Antigens Ov-103 and Ov-RAL-2 against Onchocerca volvulus in Mice.

BACKGROUND: In some regions in Africa, elimination of onchocerciasis may be possible with mass drug administration, although there is concern based on several factors that onchocerciasis cannot be eliminated solely through this approach. A vaccine against Onchocerca volvulus would provide a critical tool for the ultimate elimination of this infection. Previous studies have demonstrated that immunization of mice with Ov-103 and Ov-RAL-2, when formulated with alum, induced protective immunity. It was hypothesized that the levels of protective immunity induced with the two recombinant antigens formulated with alum would be improved by formulation with other adjuvants known to enhance different types of antigen-specific immune responses. METHODOLOGY/ PRINCIPAL FINDINGS: Immunizing mice with Ov-103 and Ov-RAL-2 in conjunction with alum, Advax 2 and MF59 induced significant levels of larval killing and host protection. The immune response was biased towards Th2 with all three of the adjuvants, with IgG1 the dominant antibody. Improved larval killing and host protection was observed in mice immunized with co-administered Ov-103 and Ov-RAL-2 in conjunction with each of the three adjuvants as compared to single immunizations. Antigen-specific antibody titers were significantly increased in mice immunized concurrently with the two antigens. Based on chemokine levels, it appears that neutrophils and eosinophils participate in the protective immune response induced by Ov-103, and macrophages and neutrophils participate in immunity induced by Ov-RAL-2. CONCLUSIONS/SIGNIFICANCE: The mechanism of protective immunity induced by Ov-103 and Ov-RAL-2, with the adjuvants alum, Advax 2 and MF59, appears to be multifactorial with roles for cytokines, chemokines, antibody and specific effector cells. The vaccines developed in this study have the potential of reducing the morbidity associated with onchocerciasis in humans

Fold differences in mean cytokine levels from mice immunized with co-administration of <i>Ov-</i>103 and <i>Ov-</i>RAL-2 with adjuvant.

<p>Fold differences in mean cytokine levels from mice immunized with co-administration of <i>Ov-</i>103 and <i>Ov-</i>RAL-2 with adjuvant.</p

Fold differences in mean chemokine levels from mice immunized with co-administration of <i>Ov-</i>103 and <i>Ov-</i>RAL-2 with adjuvant.

<p>Fold differences in mean chemokine levels from mice immunized with co-administration of <i>Ov-</i>103 and <i>Ov-</i>RAL-2 with adjuvant.</p

Survival of <i>Onchocerca volvulus</i> in mice immunized with <i>Ov-</i>103 with five different adjuvants.

<p>Effect of immunization with <i>Ov-</i>103 formulated with the adjuvants alum, Advax 1, Advax 2, CpG or MF59 on the development of protective immunity to <i>Onchocerca volvulus</i> larvae in mice. Each dot represents percent larval recovery from an individual animal. Data lines presented are mean ± standard error. Asterisk represents statistical difference in larval recoveries, p value ≤ 0.05. Dotted line is placed at the 95^th confidence interval for parasite recovery from control animals. % Reduction = percent reduction in parasite survival in immunized mice as compared to controls. Host Protection = percentage of mice in the immunized group with parasite recovery levels below the 95^thconfidence interval for parasite recovery from control animals.</p

Survival of <i>Onchocerca volvulus</i> in mice immunized with <i>Ov-</i>103 and <i>Ov-</i>RAL-2 co-administered with three different adjuvants.

<p>Effect of immunization with the <i>Ov-</i>103 and <i>Ov-</i>RAL-2 co-administered vaccines formulated with the adjuvants alum, Advax 2 or MF59 on the development of protective immunity to <i>Onchocerca volvulus</i> larvae in mice. Each dot represents percent larval recovery from an individual animal. Data lines presented are mean ± standard error. Asterisk represents statistical difference in larval recoveries, p value ≤ 0.05. Dotted line is placed at the 95^th confidence interval for parasite recovery from control animals. % Reduction = percent reduction in parasite survival in immunized mice as compared to controls. Host Protection = percentage of mice in the immunized group with parasite recovery levels below the 95^thconfidence interval for parasite recovery from control animals.</p

Survival of <i>Onchocerca volvulus</i> in mice immunized with <i>Ov-</i>RAL-2 with three different adjuvants.

<p>Effect of immunization with <i>Ov-</i>RAL-2 formulated with the adjuvants alum, Advax 2 or MF59 on the development of protective immunity to <i>Onchocerca volvulus</i> larvae in mice. Each dot represents percent larval recovery from an individual animal. Data lines presented are mean ± standard error. Asterisk represents statistical difference in larval recoveries, p value ≤ 0.05. Dotted line is placed at the 95^th confidence interval for parasite recovery from control animals. % Reduction = percent reduction in parasite survival in immunized mice as compared to controls. Host Protection = percentage of mice in the immunized group with parasite recovery levels below the 95^thconfidence interval for parasite recovery from control animals.</p

Cytokines and chemokines measured by Luminex.

<p>Cytokines and chemokines measured by Luminex.</p

Survival of <i>Onchocerca volvulus</i> in mice immunized with <i>Ov-</i>103 or <i>Ov-</i>RAL-2 without adjuvant.

<p>Effect of immunization with <i>Ov-</i>103 or <i>Ov-</i>RAL-2 without adjuvant on the development of protective immunity to <i>Onchocerca volvulus</i> larvae in mice. Each dot represents percent larval recovery from an individual animal. Data presented are mean ± standard error.</p

Fold differences in mean chemokine levels from mice immunized with <i>Ov-</i>103 or <i>Ov-</i>RAL-2 with and without adjuvant.

<p>Fold differences in mean chemokine levels from mice immunized with <i>Ov-</i>103 or <i>Ov-</i>RAL-2 with and without adjuvant.</p