Immunological characterization of potential protective proteins of Mycobacterium avium subspecies Paratuberculosis

Mycobacterium avium subsp. paratuberculosis ( M. paratuberculosis), the causative agent of bovine paratuberculosis, is very closely related to M. avium subsp. avium ( M. avium). A new generation paratuberculosis vaccine that can confer better protection without causing any adverse reactions will greatly aid in the control of this disease in cattle. One approach to developing such a vaccine is to utilize bacterial, viral or DNA vaccine vectors known to stimulate robust cell-mediate immunity (CMI) for expression and delivery of protective protein(s) of M. paratuberculosis. In this study, we used Brucella abortus RB51 (RB51), replication-defective human adenovirus (HAd) and plasmid DNA (pSecTag) as vaccine vectors to deliver selected potential protective proteins of M. paratuberculosis. Based on review of the published literature, we selected four potential protective protein candidates of M. paratuberculosis: Fibronectin attachment protein (FAP), Antigen 85B (85B), Superoxide dismutase (SOD), and 35 kDa Major membrane protein (MMP). We tested the ability of the generated recombinant vaccines to induce antigen-specific immune responses and protection against M. avium TMC724 challenge in C57BL/6 or BALB/c mice. We first tested the ability of strain RB51 to stably express the selected proteins. Consistent expression of antigen 85B and SOD, but not FAP and MMP, was detected in the respective recombinant RB51 strains. Mice vaccinated with the recombinant RB51 strain expressing antigen 85B (RB51/85B) developed antigen-specific Th1 type CMI, but not antibodies, as determined by the production of interferon-γ, but not IL-5 and IL-4, by the in vitro stimulated splenocytes. Mice vaccinated with the recombinant RB51 strain expressing SOD (RB51/SOD) developed antigen-specific IgG2c, but not IgG1, antibodies, suggesting the development of a Th1 type immune response. However, no SOD-specific T cell responses were detected in these mice. Mice immunized with RB51/85B, but not RB51/SOD, developed protection against M. avium challenge as revealed by a significant decrease in bacterial load in their spleen. We generated two recombinant HAd that expressed antigen 85B (HAd/85B) and SOD (HAd/SOD) upon transduction of the host cells. Mice inoculated with HAd/85B and HAd/SOD developed robust antigen-specific Th1 type of immune response as determined by the IgG subisotype analyses and production of interferon-γ, but not IL-5 and IL-4, cytokines by the in vitro stimulated splenocytes. However, mice vaccinated with either HAd/85B or HAd/SOD did not exhibit increased resistance against the M. avium challenge. The protective potential of FAP and MMP were tested by constructing DNA vaccines using the eukaryotic expression plasmid pSecTag2. Immunization of BALB/c mice with the DNA vaccines expressing FAP (pSecTag/FAP) and MMP (pSecTag/MMP) induced a mixed Th1 and Th2 type of immune response and did not confer protection against M. avium challenge. Overall, these results suggest that RB51 and HAd are suitable vectors for inducing Th1 type immune responses against M. paratuberculosis proteins. Our results also indicate that not all proteins of M. paratuberculosis can be stably expressed in strain RB51 and not all expressed proteins can elicit similar extent of antigen-specific T cell responses. Nonetheless, the results of these studies support further testing of RB51/85B as a potential vaccine candidate against Johne\u27s disease in the target ruminant hosts

Establishment of Early Endpoints in Mouse Total-Body Irradiation Model.

Acute radiation sickness (ARS) following exposure to ionizing irradiation is characterized by radiation-induced multiorgan dysfunction/failure that refers to progressive dysfunction of two or more organ systems, the etiological agent being radiation damage to cells and tissues over time. Radiation sensitivity data on humans and animals has made it possible to describe the signs associated with ARS. A mouse model of total-body irradiation (TBI) has previously been developed that represents the likely scenario of exposure in the human population. Herein, we present the Mouse Intervention Scoring System (MISS) developed at the Veterinary Sciences Department (VSD) of the Armed Forces Radiobiology Research Institute (AFRRI) to identify moribund mice and decrease the numbers of mice found dead, which is therefore a more humane refinement to death as the endpoint. Survival rates were compared to changes in body weights and temperatures in the mouse (CD2F1 male) TBI model (6-14 Gy, 60Co γ-rays at 0.6 Gy min-1), which informed improvements to the Scoring System. Individual tracking of animals via implanted microchips allowed for assessment of criteria based on individuals rather than by group averages. From a total of 132 mice (92 irradiated), 51 mice were euthanized versus only four mice that were found dead (7% of non-survivors). In this case, all four mice were found dead after overnight periods between observations. Weight loss alone was indicative of imminent succumbing to radiation injury, however mice did not always become moribund within 24 hours while having weight loss >30%. Only one survivor had a weight loss of greater than 30%. Temperature significantly dropped only 2-4 days before death/euthanasia in 10 and 14 Gy animals. The score system demonstrates a significant refinement as compared to using subjective assessment of morbidity or death as the endpoint for these survival studies

Induction of Antigen-Specific Th1-Type Immune Responses by Gamma-Irradiated Recombinant Brucella abortus RB51

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli β-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize β-galactosidase. A single intraperitoneal inoculation of mice with 10(9) CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a β-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice

The survival curves for CD2F1 mice: control (•), sham (○) and total-body irradiated to 6 Gy (▼), 8 Gy (Δ), 10 Gy (■), and 14 Gy (□) with ⁶⁰Co γ-rays (0.6 Gy min^-1).

<p>Survival was monitored for 30 days post-TBI; n = 20-26/dose-group.</p

Weight loss percentage in individual mice after total-body irradiation to 8, 10, or 14 Gy with ⁶⁰Co γ-rays (0.6 Gy min^-1) over a 30-d monitoring period.

<p>Mice were observed up to 4 times daily and were humanely euthanized according to the Mouse Intervention Score System criteria (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161079#pone.0161079.t001" target="_blank">Table 1</a>). Panel A (8 Gy) shows five mice losing over 20% body weight with one of the five mice surviving for the full 30 days. Panel B (10 Gy) shows all mice losing at least 20% (d10 average), 23 losing over 25% (d11 average), and 20 losing over 30% (d12 average) body weight. These mice survived, on average, an additional 5, 2.5 and <1 days respectively after reaching these criteria with one mouse losing over 30% body weight and surviving for the full 30 days. Panel C (14 Gy) shows all mice losing over 30% body weight. On average, mice lost 20% by day 3.5, 25% by d4, and 30% by d6. These mice survived, on average, additional 4.5, 4, and 3 days respectively after reaching these criteria. * Potentially erroneous data due to measurement errors.</p

Recommended Mouse Intervention Scoring System (MISS 3).

<p>Recommended Mouse Intervention Scoring System (MISS 3).</p

Differences between MISS1 and MISS2.

<p>Differences between MISS1 and MISS2.</p

Time-dependent changes in body temperature and percentage difference compared to initial level (-d3) in sham- (A), 8Gy (B), 10Gy (C), and 14-Gy (D) groups of mice over a 30-day monitoring period.

<p>The symbols represent individual temperature (<b>□</b>) and percentage difference (▽), lines represent the mean values for given number of CD2F1 mice per group and monitoring day.</p