African Swine Fever Modified Live Vaccine Candidates: Transitioning from Discovery to Product Development through Harmonized Standards and Guidelines.

The recent centennial anniversary of R.E. Montgomery's seminal published description of "a form of swine fever" disease transmitted from wild African pigs to European domestic pigs is a call to action to accelerate African Swine Fever (ASF) vaccine research and development. ASF modified live virus (MLV) first-generation gene deleted vaccine candidates currently offer the most promise to meet international and national guidelines and regulatory requirements for veterinary product licensure and market authorization. A major, rate-limiting impediment to the acceleration of current as well as future vaccine candidates into regulatory development is the absence of internationally harmonized standards for assessing vaccine purity, potency, safety, and efficacy. This review summarizes the asymmetrical landscape of peer-reviewed published literature on ASF MLV vaccine approaches and lead candidates, primarily studied to date in the research laboratory in proof-of-concept or early feasibility clinical safety and efficacy studies. Initial recommendations are offered toward eventual consensus of international harmonized guidelines and standards for ASF MLV vaccine purity, potency, safety, and efficacy. To help ensure the successful regulatory development and approval of ASF MLV first generation vaccines by national regulatory associated government agencies, the World Organisation for Animal Health (WOAH) establishment and publication of harmonized international guidelines is paramount.

Benefit of 5 years of enzyme replacement therapy in advanced late onset Pompe. A case report of misdiagnosis for three decades with acute respiratory failure at presentation.

We report on a 57 year old female patient who presented in acute respiratory failure with severe generalized weakness. She was previously misdiagnosed for over three decades as polymyositis. She was treated with enzyme replacement therapy (ERT) for over five years, after being diagnosed with late onset Pompe Disease (LOPD). She returned to independent living with the use of non invasive ventilation at nights. ERT should be considered in the management of patients with advanced LOPD and the effects of ERT closely monitored.

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus.

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses.

Challenges and Opportunities in the Use of High and Maximum Biocontainment Facilities in Developing and Licensing Risk Group 3 and Risk Group 4 Agent Veterinary Vaccines.

New solutions are necessary for the singular global health security threat formed by endemic, epidemic, and emerging/re-emerging zoonoses, coupled with epizootic and enzootic transboundary animal diseases (TADs). This One Health issue is related to the daily interactions between wildlife, domesticated and indigenous livestock, and humans primarily associated with global trade, transboundary co-movement of humans and diverse livestock/livestock products, and agriculture production intensification and penetration into previously uninhabited areas. The World Health Organization defines Risk Group 3 (RG-3) and RG-4 pathogens as mainly viruses but also bacteria that serve as the foundation for approximately 60% of emerging infectious diseases that are zoonoses. The World Organisation for Animal Health defines trade-notifiable TADs, and subsets of these are zoonotic. Livestock vaccination policies mainly focus on TADs that are promulgated by the United Nations Food and Agriculture Organization and government agriculture agencies. The development, licensure, and product manufacturing of next-generation molecular-based RG-3 and RG-4 veterinary vaccines largely ignored by the global animal health biopharmaceutical sector can have an important positive impact on food security and One Health. There have been sharp increases in the global demand for livestock meat and milk products, especially in low- and middle-income countries in Africa and Asia. This relatively recent market driver-coupled with scientific advances in human EID and zoonotic disease vaccine platform technologies and increases in the number of high (US biosafety level 3 agriculture) and maximum (US animal biosafety level 4) biocontainment facilities with supporting workforce capabilities-offers new investment opportunities to the animal health biopharmaceutical sector. Moreover, a growing number of One Health public-private partnerships have moved the net present value calculus in favor of the financial feasibility of RG-3 and RG-4 veterinary vaccine product development and licensure. This article highlights the challenges and opportunities in the use of high and maximum biocontainment facilities in developing and licensing RG-3 and RG-4 veterinary vaccines that are safe and effective against epizootic and enzootic TADs and zoonotic diseases.

Detection of African swine fever virus utilizing the portable MatMaCorp ASF detection system.

African swine fever (ASF) has emerged as a major threat to domestic and wild suid populations, and its continued spread threatens commercial swine production worldwide. The causative agent of ASF, African swine fever virus (ASFV), possesses a linear, double stranded DNA genome. Traditional detection of ASFV relies on laboratory-based virus isolation or real-time PCR of samples, typically blood or spleen, obtained from suspect cases. While effective, these methodologies are not easily field deployable, a major limitation during disease outbreak and response management scenarios. In this report, we evaluated the MatMaCorp Solas 8® ASFV detection system, a field deployable DNA extraction and fluorescent detection device, for its ability to extract and detect ASFV from multiple sample types obtained from domestic swine experimentally infected with ASFV strain Georgia. We found that the MatMaCorp Solas 8® ASFV detection device, and affiliated MagicTip™ DNA extraction and C-SAND™ assay kits, readily detected ASFV in blood and spleen, as well as other sample types, including pinna, liver, skin, muscle and bone marrow.

Evaluation of modified Vaccinia Ankara-based vaccines against foot-and-mouth disease serotype A24 in cattle.

To prepare foot-and-mouth disease (FMD) recombinant vaccines in response to newly emerging FMD virus (FMDV) field strains, we evaluated Modified Vaccinia virus Ankara-Bavarian Nordic (MVA-BN®) as an FMD vaccine vector platform. The MVA-BN vector has the capacity to carry and express numerous foreign genes and thereby has the potential to encode antigens from multiple FMDV strains. Moreover, this vector has an extensive safety record in humans. All MVA-BN-FMD constructs expressed the FMDV A24 Cruzeiro P1 capsid polyprotein as antigen and the FMDV 3C protease required for processing of the polyprotein. Because the FMDV wild-type 3C protease is detrimental to mammalian cells, one of four FMDV 3C protease variants were utilized: wild-type, or one of three previously reported mutants intended to dampen protease activity (C142T, C142L) or to increase specificity and thereby reduce adverse effects (L127P). These 3C coding sequences were expressed under the control of different promoters selected to reduce 3C protease expression. Four MVA-BN-FMD constructs were evaluated in vitro for acceptable vector stability, FMDV P1 polyprotein expression, processing, and the potential for vaccine scale-up production. Two MVA-BN FMD constructs met the in vitro selection criteria to qualify for clinical studies: MVA-mBN360B (carrying a C142T mutant 3C protease and an HIV frameshift for reduced expression) and MVA-mBN386B (carrying a L127P mutant 3C protease). Both vaccines were safe in cattle and elicited low to moderate serum neutralization titers to FMDV following multiple dose administrations. Following FMDV homologous challenge, both vaccines conferred 100% protection against clinical FMD and viremia using single dose or prime-boost immunization regimens. The MVA-BN FMD vaccine platform was capable of differentiating infected from vaccinated animals (DIVA). The demonstration of the successful application of MVA-BN as an FMD vaccine vector provides a platform for further FMD vaccine development against more epidemiologically relevant FMDV strains.

Duration of protection and humoral immunity induced by an adenovirus-vectored subunit vaccine for foot-and-mouth disease (FMD) in Holstein steers.

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven hooved animals that continues to cause economic disruption in both endemic countries or when introduced into a formally FMD free country. Vaccines that protect against clinical disease and virus shedding are critical to control FMD. The replication deficient human adenovirus serotype 5 (Ad5) vaccine vector expressing empty FMD virus (FMDV) capsid, AdtFMD, is a promising new vaccine platform. With no shedding or spreading of viral vector detected in field trials, this vaccine is very safe to manufacture, as there is no requirement for high containment faciitites. Here, we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9 months following a single vaccination with the commercial AdtFMD vaccine, provisionally licensed for cattle in the United States. Further, we tested the effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome and humoral immunity. Results demonstrate that a single dose vaccination in cattle with the commercial vaccine vector expressing capsid proteins of the FMDV strain A24 Cruzeiro (Adt.A24), induced protection against clinical FMD at 6 months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9 months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Anti-FMDV antibody secreting cells are detected in peripheral blood early following vaccination, but are absent after 28 days post-vaccination. Thus, the decay in antibody mediated immunity over time is likely a function of FMDV-specific antibody half-life. These data reveal the short time span of anti-FMDV antibody secreting cells (ASCs) and important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.

Plant-made E2 glycoprotein single-dose vaccine protects pigs against classical swine fever.

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at-risk animals, often at very high cost. Current CSFV-modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first-generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide-spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium-mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil-in-water emulsion adjuvants. We report the manufacturing of adjuvanted, plant-made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single-dose vaccination, which was accompanied by strong virus neutralization antibody responses.

Versatility of the adenovirus-vectored foot-and-mouth disease vaccine platform across multiple foot-and-mouth disease virus serotypes and topotypes using a vaccine dose representative of the AdtA24 conditionally licensed vaccine.

We investigated the serotype- and topotype versatility of a replication-deficient human adenovirus serotype 5 vectored foot-and-mouth disease (FMD) vaccine platform (AdtFMD). Sixteen AdtFMD recombinant subunit monovalent vaccines targeting twelve distinct FMD virus (FMDV) serotype/topotypes in FMD Regional Pools I-VII were constructed. The AdtA24 serotype conditionally licensed vaccine served as the basis for vaccine design and target dose for cattle clinical trials. Several vaccines contained an additional RGD motif genetic insertion in the adenovector fiber knob, and/or a full-length 2B gene insertion in the FMDV P1 gene cassette. In 13 of the 22 efficacy studies conducted, naïve control and AdtFMD vaccinated cattle were challenged intradermolingually at 2 weeks post-vaccination using a FMDV strain homologous to the AdtFMD vaccine strain. Each of the 16 AdtFMD vaccines were immunogenic based on the presence of homologous neutralizing antibodies in the serum of approximately 90% of total vaccinates (n = 375) on the day of challenge. Importantly, for 75% of vaccines tested, the effective dose that conferred 100% protection against clinical FMD was identical to or in some cases lower than, the minimum protective dose for the conditionally licensed AdtA24 vaccine formulated with ENABL® adjuvant. Results also confirmed the capability of the AdtFMD vaccine platform to differentiate infected from vaccinated animals (DIVA) across the five FMDV serotypes evaluated. Collectively, this comprehensive set of FMD cattle vaccine dose ranging studies highlights the serotype- and topotype versatility of the AdtFMD vaccine platform for further development, licensure, and application in FMD outbreak control and disease eradication efforts.

Efficacy of an adenovirus-vectored foot-and-mouth disease virus serotype A subunit vaccine in cattle using a direct contact transmission model.

BACKGROUND: A direct contact transmission challenge model was used to simulate natural foot-and-mouth disease virus (FMDV) spread from FMDV A24/Cruzeiro/BRA/55 infected 'seeder' steers to naïve or vaccinated steers previously immunized with a replication-deficient human adenovirus-vectored FMDV A24/Cruzeiro/BRA/55 capsid-based subunit vaccine (AdtA24). In two independent vaccine efficacy trials, AdtA24 was administered once intramuscularly in the neck 7 days prior to contact with FMDV A24/Cruzeiro/BRA/55-infected seeder steers. RESULTS: In Efficacy Study 1, we evaluated three doses of AdtA24 to estimate the 50%/90% bovine protective dose (BPD50/90) for prevention of clinical FMD. In vaccinated, contact-challenged steers, the BPD50/90 was 3.1 × 1010 / 5.5 × 1010 AdtA24 particles formulated without adjuvant. In Efficacy Study 2, steers vaccinated with 5 × 1010 AdtA24 particles, exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers, did not develop clinical FMD or transmit FMDV to other vaccinated or naïve, non-vaccinated steers. In contrast, naïve, non-vaccinated steers that were subsequently exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers developed clinical FMD and transmitted FMDV by contact to additional naïve, non-vaccinated steers. The AdtA24 vaccine differentiated infected from vaccinated animals (DIVA) because no antibodies to FMDV nonstructural proteins were detected prior to FMDV exposure. CONCLUSIONS: A single dose of the AdtA24 non-adjuvanted vaccine conferred protection against clinical FMD at 7 days post-vaccination following direct contact transmission from FMDV-infected, naïve, non-vaccinated steers. The AdtA24 vaccine was effective in preventing FMDV transmission from homologous challenged, contact-exposed, AdtA24-vaccinated, protected steers to co-mingled, susceptible steers, suggesting that the vaccine may be beneficial in reducing both the magnitude and duration of a FMDV outbreak in a commercial cattle production setting.

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20-25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent-free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Strengthening One Health Through Investments in Agricultural Preparedness.

There are links among agriculture and zoonotic diseases, transboundary diseases in domesticated and wild animals, climate patterns, and human population migrations. A natural or intentionally occurring high-consequence infectious disease ("biothreat") often has no geographic boundaries and has the potential to result in disease epidemics in humans, animals, or both. Although significant strides have been made globally in preparing for a natural or intentional introduction of an emerging and/or zoonotic disease, much remains to be accomplished. Enhancing animal health and well-being is a vital component to enable a sustainable, safe, and nutritious food supply for global food economies. This article explores the biothreat environment, its One Health interrelationship, and the significance and role of US agriculture in One Health. We provide an overview of the US Emergency Medical Countermeasure Enterprise (EMCE) and current state of veterinary and zoonotic medical countermeasures portfolio management in the US government, veterinary biologic industry, not-for-profit groups, and public-private partnerships. The highest zoonotic and epizootic threats to the US livestock industry are briefly reviewed, and currently available veterinary medical countermeasures are presented. Lastly, important gaps and priorities are identified, followed by specific recommendations to address these gaps.

Use of ENABL® adjuvant to increase the potency of an adenovirus-vectored foot-and-mouth disease virus serotype A subunit vaccine.

A foot-and-mouth disease (FMD) recombinant subunit vaccine formulated with a lipid/polymer adjuvant was evaluated in two vaccine efficacy challenge studies in steers. The vaccine active ingredient is a replication-deficient human adenovirus serotype 5 vector encoding the FMD virus (FMDV) A24/Cruzeiro/BRA/55 capsid (AdtA24). In the first study, AdtA24 formulated in ENABL® adjuvant was compared to a fourfold higher dose of AdtA24 without adjuvant. Steers vaccinated with AdtA24 + ENABL® adjuvant developed a significantly higher virus neutralizing test (VNT) antibody titer and an improved clinical response following FMDV A24/Cruzeiro/BRA/55 intradermal lingual challenge at 14 days post-vaccination (dpv) than steers vaccinated with the active ingredient alone. In the second study, vaccination with AdtA24 formulated in ENABL® at the same dose used in the first study, followed by FMDV A24/Cruzeiro/BRA/55 challenge on 7 or 14 dpv, prevented clinical FMD in all steers and conferred 90% protection against viremia. In addition, post-challenge FMDV titers in nasal samples from vaccinated steers compared to unvaccinated steers were significantly reduced. In both studies, none of the AdtA24 vaccinated steers developed antibodies to the FMDV non-structural proteins prior to challenge with FMDV, indicative of the capacity to differentiate infected from vaccinated animals (DIVA). These results demonstrate that administration of AdtA24 formulated in ENABL® adjuvant lowered the protective dose and prevented clinical FMD following exposure of vaccinated steers to virulent FMDV at 7 or 14 dpv.

Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine.

African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy.

Safety and immunogenicity of mammalian cell derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine.

A reverse vaccinology system, Vaxign, was used to identify and select a subset of five African Swine Fever (ASF) antigens that were successfully purified from human embryonic kidney 293 (HEK) cells and produced in Modified vaccinia virus Ankara (MVA) viral vectors. Three HEK-purified antigens [B646L (p72), E183L (p54), and O61R (p12)], and three MVA-vectored antigens [B646L, EP153R, and EP402R (CD2v)] were evaluated using a prime-boost immunization regimen swine safety and immunogenicity study. Antibody responses were detected in pigs following prime-boost immunization four weeks apart with the HEK-293-purified p72, p54, and p12 antigens. Notably, sera from the vaccinees were positive by immunofluorescence on ASFV (Georgia 2007/1)-infected primary macrophages. Although MVA-vectored p72, CD2v, and EP153R failed to induce antibody responses, interferon-gamma (IFN-γ+) spot forming cell responses against all three antigens were detected one week post-boost. The highest IFN-γ+ spot forming cell responses were detected against p72 in pigs primed with MVA-p72 and boosted with the recombinant p72. Antigen-specific (p12, p72, CD2v, and EP153R) T-cell proliferative responses were also detected post-boost. Collectively, these results are the first demonstration that ASFV subunit antigens purified from mammalian cells or expressed in MVA vectors are safe and can induce ASFV-specific antibody and T-cell responses following a prime-boost immunization regimen in swine.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge.

The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×10(8) to 2.1×10(11) particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R(2)=0.97) and viremia (R(2)=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R(2)=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.

Outcomes of tube thoracostomies performed by advanced practice providers vs trauma surgeons.

BACKGROUND: The role of advanced registered nurse practitioners and physician assistants in emergency departments, trauma centers, and critical care is becoming more widely accepted. These personnel, collectively known as advanced practice providers, expand physicians' capabilities and are being increasingly recruited to provide care and perform invasive procedures that were previously performed exclusively by physicians. OBJECTIVES: To determine whether the quality of tube thoracostomies performed by advanced practice providers is comparable to that performed by trauma surgeons and to ascertain whether the complication rates attributable to tube thoracostomies differ on the basis of who performed the procedure. METHODS: Retrospective blinded reviews of patients' charts and radiographs were conducted to determine differences in quality indicators, complications, and outcomes of tube thoracostomies by practitioner type: trauma surgeons vs advanced practice providers. RESULTS: Differences between practitioner type in insertion complications, complications requiring additional interventions, hospital length of stay, and morbidity were not significant. The only significant difference was a complication related to placement of the tube: when the tube extended caudad, toward the feet, from the insertion site. Interrater reliability ranged from good to very good. CONCLUSIONS: Use of advanced practice providers provides consistent and quality tube thoracostomies. Employment of these practitioners may be a safe and reasonable solution for staffing trauma centers.

Parasites and immune responses: memory illusion?

Immunological memory responses to intracellular protozoa and extracellular helminths govern host resistance and susceptibility to reinfection. Humans and livestock living in parasitic disease endemic regions face continuous exposure from a very early age that often leads to asymptomatic chronic infection over their entire lifespan. Fundamental immunological studies suggest that the generation of T-cell memory is driven by tightly coordinated innate and adaptive cellular immune responses rapidly triggered following initial host infection. A key distinguishing feature of immune memory maintenance between the majority of parasitic diseases and most bacterial or viral diseases is long-term antigen persistence. Consequently, functional parasite immune memory is in a continuous, dynamic flux between activation and deactivation producing functional parasite killing or functional memory cell death. In this sense, T-cell immune memory can be regarded as "memory illusion." Furthermore, due to the finite capacity of memory lymphocytes to proliferate, continuous parasite antigen stimulation may exceed a threshold level at some point in the chronically infected host. This may result in suboptimal effector immune memory leading to host susceptibility to reinfection, or immune dysregulation yielding disease reactivation or immune pathology. The goal of this review is to highlight, through numerous examples, what is currently known about T-cell immune memory to parasites and to provide compelling hypotheses on the survival and maintenance of parasite "memory illusion." These novel concepts are discussed in the context of rationale parasite vaccine design strategies.

Immune factors influencing the course of infection with Neospora caninum in the murine host.

This paper investigates the role of specific immune factors on the course of infection in genetic knockout (gko) mice infected with 3 different strains of Neospora caninum. Survival time and parasite persistence were examined in interferon-gamma (IFN-gamma), tumor necrosis factor receptor-2 (TNFR2), interleukin 10 (IL-10), beta 2 microglobulin (beta2M), and inducible nitric oxide synthase (iNOS2) gko or wild-type (wt) mice following infection with either pathogenic (NC-1 or NC-2) or attenuated (NCts-8) N. caninum strains. Infection with NC-1 was 100% lethal in IFN-gamma gko mice, as evidenced by mean survival times of 10-13 days, depending on the challenge dose used. TNFR2 and beta2M gko mice infected with NC-1 or NC-2 strain demonstrated partial susceptibility to disease, as evidenced by histopathology and survival curves. TNFR2 or beta2M gko mice were not susceptible to infection with NCts-8, on the basis of absence of pathology and lack of mortality. Lack of mortality and minimal histopathology scores demonstrated that NC-1, NC-2, and NCts-8 infections were avirulent in IL-10 and iNOS2 gko mice. Adoptive transfer of immune cells from NCts-8 vaccinated normal syngeneic mice into IFN-gamma gko mice significantly (P < 0.05) prolonged mean survival times at all 3 challenge doses of NC-1 but failed to protect against mortality. Interestingly, there was a notable change in the tissue tropism of tachyzoites from the lung and brain in immunocompetent wt, TNFR2 gko, IL-10 gko, beta2M gko, and iNOS2 gko mice to the liver and spleen in IFN-gamma gko mice when challenged with N. caninum. On the basis of these results in gko mice, IFN-gamma is a critical cytokine in the host response against acute neosporosis.