Overview of Plasmodium spp. and Animal Models in Malaria Research.

Malaria is a parasitic disease caused by protozoan species of the genus Plasmodium and transmitted by female mosquitos of the genus Anopheles and other Culicidae. Most of the parasites of the genus Plasmodium are highly species specific with more than 200 species described affecting different species of mammals, birds, and reptiles. Plasmodium species strictly affecting humans are P. falciparum, P. vivax, P. ovale, and P. malariae. More recently, P. knowlesi and other nonhuman primate plasmodia were found to naturally infect humans. Currently, malaria occurs mostly in poor tropical and subtropical areas of the world, and in many of these countries it is the leading cause of illness and death. For more than 100 y, animal models, have played a major role in our understanding of malaria biology. Avian Plasmodium species were the first to be used as models to study human malaria. Malaria parasite biology and immunity were first studied using mainly P. gallinaceum and P. relictum. Rodent malarias, particularly P. berghei and P. yoelii, have been used extensively as models to study malaria in mammals. Several species of Plasmodium from nonhuman primates have been used as surrogate models to study human malaria immunology, pathogenesis, candidate vaccines, and treatments. Plasmodium cynomolgi, P. simiovale, and P. fieldi are important models for studying malaria produced by P. vivax and P. ovale, while P. coatneyi is used as a model for studying severe malaria. Other nonhuman primate malarias used in research are P. fragile, P. inui, P. knowlesi, P. simium, and P. brasilianum. Very few nonhuman primate species can develop an infection with human malarias. Macaques in general are resistant to infection with P. falciparum, P. vivax, P. malariae, and P. ovale. Only apes and a few species of New World monkeys can support infection with human malarias. Herein we review the most common, and some less common, avian, reptile, and mammal plasmodia species used as models to study human malaria.

Immune Effects of Cryoablation in Woodchuck Hepatocellular Carcinoma.

Objectives: Local and systemic immune responses evoked by locoregional therapies such as cryoablation are incompletely understood. The aim of this study was to characterize cryoablation-related immune response and the capacity of immune drugs to augment immunity upon cryoablation for the treatment of hepatocellular carcinoma (HCC) using a woodchuck hepatocellular carcinoma model. Materials and Methods: Twelve woodchucks chronically infected with woodchuck hepatitis virus and with hepatocellular carcinoma underwent imaging with contrast-enhanced CT. Partial cryoablation of tumors in three woodchucks was performed. Fourteen days after cryoablation, liver tissues were harvested and stained with H&E and TUNEL, and immune infiltrates were quantified. Peripheral blood mononuclear cells (PBMC) were collected from ablated and nonablated woodchucks, labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured with immune-modulating drugs, including a small PD-L1 antagonist molecule (BMS-202) and three TLR7/8 agonists (DSR 6434, GS-9620, gardiquimod). After incubation, cell replication and immune cell populations were analyzed by flow cytometry. Results: Local immune response in tumors was characterized by an increased number of CD3+ T lymphocytes and natural killer cells in the cryolesion margin compared to other tumor regions. T regulatory cells were found in higher numbers in distant tumors within the liver compared to untreated or control tumors. Cryoablation also augmented the systemic immune response as demonstrated by higher numbers of PBMC responses upon immune drug stimulation in the cryoablation group. Conclusions: Partial cryoablation augmented immune effects in both treated and remote untreated tumor microenvironments, as well as systemically, in woodchucks with HCC. Characterization of these mechanisms may enhance development of novel drug-device combinations for treatment of HCC.

Evaluation of immune-modulating drugs for use in drug-eluting microsphere transarterial embolization.

Cancer immunotherapy has yet to reach its full potential due in part to limited response rates and side effects inherent to systemic delivery of immune-modulating drugs. Local administration of immunotherapy using drug-eluting embolic (DEE) microspheres as drug delivery vehicles for direct infusion into tumor-feeding arteries might increase and prolong tumor drug concentrations and reduce systemic drug exposure, potentially improving the risk-to-benefit ratio of these agents. The purpose of this study was to evaluate the ability of four immune modulators affecting two different immune pathways to potentiate replication of immune cells from a woodchuck model of hepatocellular carcinoma. DSR 6434, a Toll-like receptor agonist, and BMS-202, a PD-L1 checkpoint inhibitor, induced immune cell replication and were successfully loaded into radiopaque DEE microspheres in high concentrations. Release of DSR 6434 from the DEE microspheres was rapid (t99% = 0.4 h) upon submersion in a physiologic saline solution while BMS-202 demonstrated a more sustained release profile (t99% = 17.9 h). These findings demonstrate the feasibility of controlled delivery of immune-modulating drugs via a local DEE microsphere delivery paradigm.

Hepatic steatosis, a lesion reported in captive aged common marmosets.

Hepatic steatosis, also known as fatty liver, is a spontaneous lesion caused by the abnormal accumulation of triglycerides within hepatocytes that has been described in different laboratory-housed nonhuman primate species. Aging is considered a risk factor in the progression of this lesion in humans and captive rhesus macaques. Hepatic steatosis has been reported in sexually mature adult and aged-adult captive common marmosets. Macroscopic changes in the liver may be evident in advanced stages of this condition and are characterized by hepatomegaly with multifocal to coalescing to regionally extensive pale-tan to yellow, soft foci throughout the hepatic lobes. Biochemical abnormalities in these cases include significantly increased levels in triglycerides, insulin, and γ-glutamyltransferase (GGT). Definitive diagnosis is by histopathology and demonstration of lipid accumulation within hepatocytes. Histopathology is characterized by large coalescing areas of periacinar to periportal microvesicular steatosis mixed with clusters of macrovesicular steatosis, and variable degrees of lobular inflammation. Vacuolated hepatocytes containing intracytoplasmic lipid material is demonstrated by positive staining to Sudan IV and/or Oil red-O.

A Combined YopB and LcrV Subunit Vaccine Elicits Protective Immunity against Yersinia Infection in Adult and Infant Mice.

Yersinia enterocolitica causes a severe enteric infection in infants and young children. There is no vaccine approved for use in humans. We investigated the immunogenicity and protective capacity of Yersinia YopB, a conserved type III secretion system protein, alone or combined with LcrV in adult mice immunized intranasally. YopB or LcrV (5 µg) administered with the Escherichia coli double mutant heat-labile toxin (dmLT) adjuvant afforded modest (10-30%) protection against lethal Y. enterocolitica oral infection. The combination of YopB and LcrV (5 µg each) dramatically improved vaccine efficacy (70-80%). Additionally, it afforded complete protection against Y. pestis pulmonary infection. Immunization with YopB/LcrV+dmLT resulted in Ag-specific serum IgG, systemic and mucosal Ab-secreting cells, as well as IFN-γ, TNF-α, IL-2, IL-6, IL-17A, and KC production by spleen cells. Serum Abs elicited by YopB/LcrV+dmLT had enhanced bactericidal and opsonophagocytic killing activity. After Y. enterocolitica challenge, YopB/LcrV+dmLT-vaccinated mice exhibited intact intestinal tissue, active germinal centers in mesenteric lymph nodes, IgG+ and IgA+ plasmablasts in the lamina propria, and Abs in intestinal fluid. On the contrary, complete tissue destruction and abscesses were seen in placebo recipients that succumbed to infection. Mice immunized as infants with YopB+dmLT or LcrV+dmLT achieved 60% protection against lethal Y. enterocolitica infection, and vaccine efficacy increased to 90-100% when they received YopB/LcrV+dmLT. YopB+dmLT also afforded substantial (60%) protection when administered intradermally to infant mice. YopB/LcrV+dmLT is a promising subunit vaccine candidate with the potential to elicit broad protection against Yersinia spp.

Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

Shigella IpaB and IpaD displayed on L. lactis bacterium-like particles induce protective immunity in adult and infant mice.

Shigella spp. are among the enteric pathogens with the highest attributable incidence of moderate-to-severe diarrhea in children under 5 years of age living in endemic areas. There are no vaccines available to prevent this disease. In this work, we investigated a new Shigella vaccine concept consisting of nonliving, self-adjuvanted, Lactococcus lactis bacterium-like particles (BLP) displaying Shigella invasion plasmid antigen (Ipa) B and IpaD and examined its immunogenicity and protective efficacy in adult and newborn/infant mice immunized via the nasal route. Unique advantages of this approach include the potential for broad protection due to the highly conserved structure of the Ipas and the safety and practicality of a probiotic-based mucosal/adjuvant delivery platform. Immunization of adult mice with BLP-IpaB and BLP-IpaD (BLP-IpaB/D) induced high levels of Ipa-specific serum IgG and stool IgA in a dose-dependent manner. Immune responses and protection were enhanced by BLP delivery. Vaccine-induced serum antibodies exhibited opsonophagocytic and cytotoxic neutralizing activity, and IpaB/D IgG titers correlated with increased survival post-challenge. Ipa-specific antibody secreting cells were detected in nasal tissue and lungs, as well as IgG in bronchoalveolar lavage. Bone marrow cells produced IpaB/D-specific antibodies and contributed to protection after adoptive transfer. The BLP-IpaB/D vaccine conferred 90% and 80% protection against S. flexneri and S. sonnei, respectively. Mice immunized with BLP-IpaB/D as newborns also developed IpaB and IpaD serum antibodies; 90% were protected against S. flexneri and 44% against S. sonnei. The BLP-IpaB/D vaccine is a promising candidate for safe, practical and potentially effective immunization of children against shigellosis.

Development and preliminary validation of an antibody filtration-assisted single-dilution chemiluminometric immunoassay for potency testing of Piscirickettsia salmonis vaccines.

Challenge with live pathogens could be substituted by serology for many veterinary diseases, however little progress has been made in the development of alternative batch vaccine potency tests for fish. This study reports the development and preliminary validation of a single-dilution filtration-assisted chemiluminometric immunoassay (SD FAL-ELISA) applied to measure anti Piscirickettsia salmonis IgM in individual or pooled serum and mucus samples. The assay was set up to test a single-dilution of the sample. Serum SD FAL-ELISA yielded a sensitivity of 90% and a specificity of 96%. SD FAL-ELISA was applied to evaluate pooled and individual samples from P. salmonis challenge assessments. Relative-light units values (RLU) obtained by SD FAL-ELISA were proportional to antibody levels in serum. RLU values obtained from pooled and individual serum samples increased with the observed relative percent survival (RPS) values, indicating a correlation between protection and specific IgM levels. Results obtained for specific IgM in mucus samples was not related to the RPS, but discriminated the vaccine that yielded high RPS (86.4%) from the others (40.9 and 54.5%). This is the first report on the development of an indirect high-throughput serological assessment for P. salmonis vaccine potency testing using both pooled or individual serum and cutaneous mucus samples.

Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle.

Serological assessment of the heterologous response among Foot-and-Mouth Disease Virus (FMDV) strains is mainly performed by virus neutralization test (VNT), liquid phase blocking ELISA and complement fixation assay. In this study two high-throughput ELISA techniques, avidity and IgG subtype ELISA, were developed and used to further characterize heterologous antibody responses in cattle during vaccination and challenge. Both assays were applied to a set of previously characterized sera from animals immunized with an inactivated A24 Cruzeiro/Brazil/55 (A24 Cruzeiro) strain monovalent FMDV vaccine and challenged with the heterologous A/Argentina/2001 (A/Arg/01) strain. Single dilution avidity ELISA assessment showed that animals that were protected against A/Arg/01 challenge had higher avidity antibodies to this heterologous strain than non-protected cattle. Animals with low or even undetectable anti-A/Arg/01 serum-neutralizing titers that passed the heterologous challenge presented higher IgG1/IgG2 ratio than non-protected animals. In this study, the three assessments (VNT and both ELISAs) discriminated between protected and not protected animals against a heterologous challenge. The combination of these techniques may be applied to complement current indirect serological vaccine-matching assessments. The measurement of these qualitative parameters may provide additional information to understand the mechanisms underlying FMD heterologous responses and the induction of cross-protection in cattle.

The immune enhancement of a novel soy lecithin/ß-glucans based adjuvant on native Neospora caninum tachyzoite extract vaccine in mice.

Efficient, cost-effective and safe Th1-immunity-inducing vaccine formulations are paramount for achieving protection against Neospora caninum. In this study, a new adjuvant (Providean-AVEC) was used in the development of a N. caninum vaccine and evaluated in a mouse model. Soluble N. caninum tachyzoite native protein extract (sNcAg) was selected as vaccine antigen based on its capacity to activate production of pro-inflammatory cytokines on dendritic cells. Vaccines containing 4 and 0.4 µg of sNcAg, and Providean-AVEC, ISCOM-Matrix or aluminum hydroxide (Alum) were tested in BALB/c mice. While mice vaccinated with 4µg of sNcAg + Providean-AVEC developed specific antibodies shortly after the first dose, the rest of the high antigen payload formulations only induced seroconversion after the booster. Mice immunized with the high payload ISCOM vaccine (4 µg sNcAg) or with either low or high payload Providean-AVEC formulations (0.4 µg and 4 µg sNcAg, respectively) elicited higher IgG2a than IgG1 serum levels, and IFN-γ anamnestic responses with a Th1-cytokine biased profile. These animals had no histological signs of cerebral lesions and parasite burden assessed by quantitative real-time PCR was not detected. Vaccine preparations including Providean-AVEC as adjuvant limited N. canimum multiplication even with only a tenth of antigen payload compared to vaccines containing other adjuvants. Using adjuvants to specifically activate dendritic cells, combined with a careful antigen selection can enhance cellular responses to inert N. caninum vaccines.

Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle.

Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.