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Completion of a 5-day course of remdesivir was associated with approximately 17-fold increased odds of survival among a sample of 54 nursing home residents with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the course of an outbreak from October to December 2020. Remdesivir was well tolerated; administration was logistically feasible in a pre-hospital environment.

Infrared thermometry (IRTM) is a noncontact method to measure temperature. The purpose of this study was to compare rectal temperature and IRTM in healthy anesthetized swine, with the hypothesis that IRTM would be an accurate, noninvasive alternative for rectal temperature measurement. Two groups of female Yorkshire-cross swine (n = 14 and n = 12) were sedated with Tiletamine–zolazepam (0.5 mg/kg) for blood collection during a routine physical examination. While sedated, rectal temperatures were measured using a SureTemp Plus 690 (Welch Allyn) and IRTM measurements were taken using a FLIR E5 thermal imaging camera. The 2 anatomic sites used for thermography measurements were the area surrounding the eye and the neck at the base of the ear. The distance from the imaging camera and the animal during IRTM measurements was 24 to 32 inches, a distance that would allow camera access in a standard swine enclosure. The infrared imaging camera's surface temperature measurement exhibited a proportional bias when compared with the rectal temperature. All rectal temperature measurements were between 98.7 °F to 101.3 °F, with a mean temperature of 100.4 °F. IRTM tended to underestimate rectal temperatures at lower values, and overestimate rectal temperatures at higher values by approximately (+) or (-) 0.8 °F of rectal temperature. Infrared thermometry can provide a quick noninvasive assessment of the body surface temperature, without the need for animal handling or restraint, but should not be considered an accurate replacement for rectal temperature measurement.

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease.

Following vaccination with the live attenuated, recombinant vesicular stomatitis virus Indiana serotype Ebola virus (rVSV-EBOV) vaccine, persons may exhibit a transient vaccine-associated viremia. To investigate the potential for Old World sand flies to transmit this vaccine following feeding on a viremic person, we fed laboratory-reared Phlebotomus papatasi an artificial blood meal containing 7.2 log10 plaque-forming units of rVSV-EBOV. Replication or dissemination was not detected in the body or legs of any P. papatasi collected at seven (n = 75) or 15 (n = 75) days post-feed. These results indicate a low potential for rVSV-EBOV to replicate and disseminate in P. papatasi, a species whose geographic distribution ranges from Morocco to southwest Asia and as far north as southern Europe.

Background Western Equine Encephalitis (WEE) is a naturally acquired infection and potentially devastating bioweapon, with no specific human countermeasures. An experimental inactivated Western Equine Encephalitis Vaccine (WEEV; WEE TSI-GSD 210) has been used under an IND (investigational New Drug) protocol at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 1976. Methods Over 24 years from 1987 to 2011, 876 subjects received 3 primary vaccine doses under 3 studies with 1,537 booster doses administered (FY87-8, phase 2, laboratory workers, vaccine lots 1-81-1, 1-81-2, and 2-1-91; FY99-12, phase 2 laboratory workers, lot 2-1-91; and FY09-02, phase 1 healthy volunteer, lot 3-1-92). Post-vaccination safety and immunogenicity [plaque reduction neutralization test 80% (PRNT80) > 1:40] were analyzed. Results Overall PRNT80 response to the primary series in FY87-8 was 42% (326/770) but dropped to 16% (14/87) in FY99-12, prompting study FY09-02, which achieved 89% (17/19). The first booster response rate was 68% (814/1194) in FY87-8, 53% (171/324) in FY99-12, and 100% (10/10) in FY09-02. The majority of definitely related adverse reactions (AEs) were mild and local with no definitely related serious AEs. No laboratory acquired WEE infection was documented during this period despite 4 reported exposures in vaccinated subjects. Conclusion The TSI-GSD 210 WEE vaccine was immunogenic, safe and well tolerated. Use of this vaccine could be considered in an emergency setting. Despite decades of safe and effective use under IND, full licensure is not planned due to manufacturing constraints, and a strategic decision to develop alternatives. Clinical trial registration https://clinicaltrials.gov/, identifier NCT01159561.

BACKGROUND Eastern equine encephalitis virus (EEEV) is a mosquito borne alphavirus spread primarily in Atlantic and Gulf Coast regions of the United States. EEEV is the causative agent of a devastating meningoencephalitis syndrome, with approximately 30% mortality and significant morbidity. There is no licensed human vaccine against EEEV. An inactivated EEEV vaccine has been offered under investigational new drug (IND) protocols at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 1976. METHODS Healthy at-risk laboratory personnel received inactivated PE-6 strain EEEV (TSI-GSD 104) vaccine under two separate IND protocols. Protocol FY 99-11 (2002-2008) had a primary series consisting of doses on day 0, 7, and 28. Protocol FY 06-31 (2008-2016) utilized a primary series with doses on day 0 and 28, and month 6. Participants with an inadequate immune response, plaque reduction neutralization test with 80% cut-off (PRNT80) titer

To evaluate potential immunocompetent small animal models of Zika virus (ZIKV) infection, we inoculated Syrian golden hamsters (subcutaneously or intraperitoneally) and strain 13 guinea pigs (intraperitoneally) with Senegalese ZIKV strain ArD 41525 or Philippines ZIKV strain CPC-0740. We did not detect viremia in hamsters inoculated subcutaneously with either virus strain, although some hamsters developed virus neutralizing antibodies. However, we detected statistically significant higher viremias (P = 0.0285) and a higher median neutralization titer (P = 0.0163) in hamsters inoculated intraperitoneally with strain ArD 41525 compared with strain CPC-0740. Furthermore, some hamsters inoculated with strain ArD 41525 displayed mild signs of disease. By contrast, strain 13 guinea pigs inoculated intraperitoneally with either strain did not have detectable viremias and less than half developed virus neutralizing antibodies. Our results support the use of the Syrian golden hamster intraperitoneal model to explore phenotypic variation between ZIKV strains.

Infrared thermometry (IRTM) is a noncontact method to measure temperature. The purpose of this study was to compare rectal temperature and IRTM in healthy anesthetized swine, with the hypothesis that IRTM would be an accurate, noninvasive alternative for rectal temperature measurement. Two groups of female Yorkshire-cross swine (n = 14 and n = 12) were sedated with Tiletamine–zolazepam (0.5 mg/kg) for blood collection during a routine physical examination. While sedated, rectal temperatures were measured using a SureTemp Plus 690 (Welch Allyn) and IRTM measurements were taken using a FLIR E5 thermal imaging camera. The 2 anatomic sites used for thermography measurements were the area surrounding the eye and the neck at the base of the ear. The distance from the imaging camera and the animal during IRTM measurements was 24 to 32 inches, a distance that would allow camera access in a standard swine enclosure. The infrared imaging camera's surface temperature measurement exhibited a proportional bias when compared with the rectal temperature. All rectal temperature measurements were between 98.7 °F to 101.3 °F, with a mean temperature of 100.4 °F. IRTM tended to underestimate rectal temperatures at lower values, and overestimate rectal temperatures at higher values by approximately (+) or (-) 0.8 °F of rectal temperature. Infrared thermometry can provide a quick noninvasive assessment of the body surface temperature, without the need for animal handling or restraint, but should not be considered an accurate replacement for rectal temperature measurement.

Background Eastern equine encephalitis virus (EEEV) is an alphavirus with a high mortality rate and serious neurological sequelae in infected persons making this virus an important human pathogen. Methods Following written informed consent, eligible subjects received two priming doses of EEEV vaccine, inactivated, TSI-GSD 104, 0.5 mL subcutaneously on days 0 and 28 days followed by a mandatory booster, 0.1 mL intradermal, at 6 months. Serum samples were collected pre-vaccination, days 21–35 following dose 2, as well as before and 21–35 days after dose 3. Sera with a Plaque Reduction Neutralization Test80 titer (PRNT80) ≥ 1:40 were considered responders with adequate titers for the purpose of biocontainment suite entry. Results Sixty-seven (67) subjects were enrolled in this study to receive the primary vaccination series. All 67 subjects received at least 1 primary vaccination; 66 completed the 2 primary doses; 58 completed the 2 primary doses and the 6-month dose. Of these, 38 (56.7%) reported one or more adverse events. Fatigue was reported in 13 (19.4%), headache in 9 (13.4%), upper respiratory tract infection in 6 (9.0%), nausea in 5 (7.5%), pyrexia in 5 (7.5%), oropharyngeal pain in 4 (6.0%), myalgia in 4 (6.0%), injection site pain in 7 (10.4%), injection site hematoma in 4 (6.0%) and injection site erythema in 3 (4.5%) subjects. Adverse events were mostly mild or moderate and transient. PRNT80 titers ≥ 1:40 was observed in 39/65 (60%) subjects who received both primary doses of EEEV vaccine compared with 48/57 (84%) subjects who completed the 2-dose primary series and the 6-month dose and also had blood drawn for titer. Females had a higher response rate (61.5%) at the pre 6-month boost titer than did males (34.3%) (p = 0.0231). Similarly, the pre 6-month boost geometric mean titer (GMT) for females was 35.5 vs. 21.9 for males (p = 0.0231). The post 6-month boost GMT for females was 146.7 and 181.5 for males (P = 0.13). Conclusion Inactivated Eastern Equine Encephalitis Virus vaccine, TSI-GSD 104, Lot 2-1-89 appears to be safe and immunogenic. This Phase 2 vaccine study supports a priming dose schedule of Days 0 and 28 and 6-month. The 6 month dose is anamnestic improving the overall response rate and level of antibody for this primary dosing schedule. Disclosures All authors: No reported disclosures.

Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development., Highlights • Adjuvants can prolong the protection afforded by protein-based vaccines and impact adaptive immune responses • Enhanced CD4 T cell responses, helper and effector, correlate with duration of protection • Durable protection from ma-EBOV is associated with Tfh frequency, Th1 antibody titers, and effector CD4 T cells Protein-based vaccines are extremely safe, but they sometimes require the addition of adjuvants to enhance immunogenicity. In this study, we compared the impact of multiple adjuvants on immunogenicity, focusing on the duration of vaccine-mediated protection in mice. We then looked at how each adjuvant impacted the immune response in order to identify correlates of that long lasting immunity. The most effective adjuvant/vaccine combinations elicited multifunctional CD4 T cell responses and a Th1-skewed antibody response. By transferring antigen-experienced CD4 T cells and serum into naïve animals, we demonstrated that both CD4 T cells and serum were critical for durable vaccine-mediated protection.

Ricin is a potent toxin and potential bioterrorism weapon for which no specific licensed countermeasures are available. We report the safety and immunogenicity of the ricin vaccine RVEc™ in a Phase 1 (N=30) multiple-dose, open-label, non-placebo-controlled, dose-escalating (20, 50, and 100μg), single-center study. Each subject in the 20- and 50-μg dose groups (n=10 for each group) received three injections at 4-week intervals and was observed carefully for untoward effects of the vaccine; blood was drawn at predetermined intervals after each dose for up to 1 year. RVEc™ was safe and well tolerated at the 20- and 50-μg doses. The most common adverse events were pain at the injection site and headache. Of the 10 subjects who received a single 100-μg dose, two developed elevated creatine phosphokinase levels, which resolved without sequelae. No additional doses were administered to subjects in the 100-μg group. Immunogenicity of the vaccine was evaluated by measuring antibody response using the well standardized enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). Of the subjects in the 20- and 50-μg dose groups, 100% achieved ELISA anti-ricin IgG titers of 1:500 to 1:121,500 and 50% produced neutralizing anti-ricin antibodies measurable by TNA. Four subjects in the 50-μg group received a single booster dose of RVEc™ 20-21 months after the initial dose. The single booster was safe and well tolerated, resulting in no serious adverse events, and significantly enhanced immunogenicity of the vaccine in human subjects. Each booster recipient developed a robust anamnestic response with ELISA anti-ricin IgG titers of 1:13,500 to 1:121,500 and neutralizing antibody titers of 1:400 to 1:3200. Future studies will attempt to optimize dose, scheduling, and route of administration. This study is registered at clinicaltrials.gov (NCT01317667 and NCT01846104).

Multiple products are being developed for use against filoviral infections. Efficacy for these products will likely be demonstrated in nonhuman primate models of filoviral disease to satisfy licensure requirements under the Animal Rule, or to supplement human data. Typically, the endpoint for efficacy assessment will be survival following challenge, however, there exists no standardized approach for assessing the health or euthanasia criteria for filovirus-exposed nonhuman primates. Consideration of objective criteria is important to (a) ensure test subjects are euthanized without unnecessary distress, (b) enhance the likelihood that animals exhibiting mild or moderate signs of disease are not prematurely euthanized, (c) minimize the occurrence of spontaneous deaths and loss of end-stage samples, (d) enhance the reproducibility of experiments between different researchers, and (e) provide a defensible rationale for euthanasia decisions that withstands regulatory scrutiny. Historic records were compiled for 58 surviving and non-surviving monkeys exposed to Ebola virus at the US Army Medical Research Institute of Infectious Diseases. Clinical pathology parameters were statistically analyzed and those exhibiting predicative value for survival are reported. These findings may be useful for standardization of objective euthanasia assessments in rhesus monkeys exposed to Ebola virus and may serve as a useful approach for other standardization efforts.

The ability to quickly and accurately determine cortisol as a biomarker for stress is a valuable tool in assessing the wellbeing of NHP. In this study, 2 methods of collecting saliva (a commercial collection device and passive drool) and the resulting free salivary cortisol levels were compared with total serum cortisol concentration in rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis) and African green monkeys (Chlorocebus aethiops) at 2 collection time points. Serum and salivary cortisol levels were determined using a competitive quantitative ELISA. In addition, both saliva collection methods were evaluated for volume collected and ease of use. Compared with passive drool, the experimental collection device was more reliable in collecting sufficient volumes of saliva, and the resulting salivary cortisol values demonstrated stronger correlation with serum cortisol concentration in all species and collection days except cynomolgus macaques on day 1. This saliva collection device allows quick and reliable sample collection for the determination of salivary cortisol levels. In addition, the results might provide a useful tool for evaluating hypothalamic-pituitary-adrenal axis activity or the physiologic stress reaction in NHP as well as a biomarker of psychologic stress states in a variety of situations.

A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter.

A recombinant fusion protein composed of Yersinia pestis fraction 1 capsule (F1) and virulence-associated V antigen (V) (F1-V) has been developed as the next-generation vaccine against plague. In this study, female Swiss Webster mice received a single intramuscular vaccination with one of eight doses of the F1-V vaccine and exposed 4 weeks later to either Y. pestis CO92 or C12 organisms by the subcutaneous or aerosol routes of infection. Quantitative anti-F1 and anti-V immunoglobulin G (IgG) ELISAs were used to examine the relationship between survival outcome and antibody titers to F1 and V. Results suggested that each 1log(10) increase in week 4 quantitative anti-F1 and anti-V IgG ELISA titers were associated with a 1.7-fold (p=0.0051) and 2.5-fold (p=0.0054) increase in odds of survival, respectively, against either bubonic or pneumonic plague and may serve as serological correlates of protection.

The current U.S. Department of Defense candidate plague vaccine is a fusion between two Yersinia pestis proteins: the F1 capsular protein, and the low calcium response (Lcr) V-protein. We hypothesized that an immunomodulator, such as CpG oligodeoxynucleotide (ODN)s, could augment the immune response to the plague F1-V vaccine in a mouse model for plague. CpG ODNs significantly augmented the antibody response and efficacy of a single dose of the plague vaccine in murine bubonic and pneumonic models of plague. In the latter study, we also found an overall significant augmentation the immune response to the individual subunits of the plague vaccine by CpG ODN 2006. In a long-term, prime-boost study, CpG ODN induced a significant early augmentation of the IgG response to the vaccine. The presence of CpG ODN induced a significant increase in the IgG2a subclass response to the vaccine up to 5 months after the boost. Our studies showed that CpG ODNs significantly augmented the IgG antibody response to the plague vaccine, which increased the probability of survival in murine models of plague ( P

In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.

Samples of human plasma from anthrax vaccine adsorbed (AVA, BioThrax™)-vaccinated individuals were used to demonstrate passive protection of A/J mice from a lethal challenge with the Sterne strain of anthrax bacteria. The maximum concentration of human anti-protective antigen IgG in mouse sera 24 h after injection of 260 μg of anti-PA IgG was 134 μg/ml, declining to 91 μg/ml at 72 h (half-life = 101.7 h). Mice showed significant survival (p ≤ 0.001) after injection of serial dilutions up to 1:4 of the standard plasma and challenged with 100 LD50. Similarly, mice injected with the standard anti-AVA plasma and challenged up to 5 days post-treatment also survived (p ≤ 0.001). Using a cohort of human plasma to measure passive protection, the best correlation between passive protection and an in vitro assay was found to be with the quantitative toxin neutralization assay (minimum fold increase in odds of survival: 2.71, p = 0.0062). These results demonstrate a reliable in vivo neutralization method that correlates with standard in vitro measures of neutralizing antibody levels in plasma from individuals vaccinated with the standard anthrax vaccine. This analytical method may provide additional opportunities to compare the efficacy of improved anthrax vaccines with the licensed vaccine.

Burkholderia pseudomallei is the causative agent of melioidosis, a bacterial infection endemic in tropical regions including southeast Asia and northern Australia. B. pseudomallei contains structurally unique polysaccharides (capsular polysaccharide and O−antigen saccharides of lipopolysaccharide). A polysaccharide microarray platform was developed by immobilizing these polysaccharides onto glass slides. Employing this microarray, we were able to demonstrate the presence of antibodies to these polysaccharide antigens in the sera of melioidosis patients, but not in serum from nonmelioidosis human subjects. The advantages of this polysaccharide microarray technology over the conventional tests for the serodiagnosis of melioidosis are discussed.

Cynomolgus macaques exposed to an aerosol containing a virulent strain of eastern equine encephalitis (EEE) virus developed neurological signs indicating encephalitis that corresponded with the onset of fever and an elevated heart rate. Viremia was either transient or undetectable even in animals that succumbed to the illness. The onset of illness was dose dependent, but once a febrile response was observed, macaques were moribund within 36 h. Simultaneously, a prominent leukocytosis was seen; 1 day before being moribund, macaques had a white blood cell count >20,000 cells/ microL. The leukocytes were predominantly granulocytes. Increases in serum levels of blood urea nitrogen, sodium, and alkaline phosphatase were also seen. The rapid onset and severity of neurological signs mirror what has been reported for human cases of disease caused by EEE.

An anthrax spore aerosol infection mouse model was developed as a first test of in vivo efficacy of antibiotics identified as active against Bacillus anthracis . Whole-body, 50% lethal dose (LD 50 ) aerosol challenge doses in a range of 1.9 × 10 3 to 3.4 × 10 4 CFU with spores of the fully virulent Ames strain were established for three inbred and one outbred mouse strain (A/J, BALB/c, C57BL, and Swiss Webster). The BALB/c strain was further developed as a model for antibiotic efficacy. Time course microbiological examinations of tissue burdens in mice after challenge showed that spores could remain dormant in the lungs while vegetative cells disseminated to the mediastinal lymph nodes and then to the spleen, accompanied by bacteremia. For antibiotic efficacy studies, BALB/c mice were challenged with 50 to 100 LD 50 of spores followed by intraperitoneal injection of either ciprofloxacin at 30 mg/kg of body weight (every 12 h [q12h]) or doxycycline at 40 mg/kg (q6h). A control group was treated with phosphate-buffered saline (PBS) q6h. Treatment was begun 24 h after challenge with groups of 10 mice for 14 or 21 days. The PBS-treated control mice all succumbed (10/10) to inhalation anthrax infection within 72 h. Sixty-day survival rates for ciprofloxacin and doxycycline-treated groups were 8/10 and 9/10, respectively, for 14-day treatment and 10/10 and 7/10 for 21-day treatment. Delayed treatment with ciprofloxacin initiated 36 and 48 h postexposure resulted in 80% survival and was statistically no different than early (24 h) postexposure treatment. Results using this mouse model correlate closely with clinical observations of inhalational anthrax in humans and with earlier antibiotic studies in the nonhuman primate inhalational anthrax model.

An outbreak or deliberate release of Rift Valley fever (RVF) virus could have serious public health and socioeconomic consequences. A safe RVF vaccine capable of eliciting long-lasting immunity after a single injection is urgently needed. The live attenuated RVF MP-12 vaccine candidate has shown promise in Phase 1 clinical trials; no evidence of reversion to virulence has been identified in numerous animal studies. The objective of this Phase 2 clinical trial was to (a) further examine the safety and immunogenicity of RVF MP-12 in RVF virus–naïve humans and (b) characterize isolates of RVF MP-12 virus recovered from the blood of vaccinated subjects to evaluate the genetic stability of MP-12 attenuation. We found that RVF MP-12 was well tolerated, causing mostly mild reactions that resolved without sequelae. Of 19 subjects, 18 (95%) and 19 (100%) achieved, respectively, 80% and 50% plaque reduction neutralization titers (PRNT80 and PRNT50) ≥ 1:20 by postvaccination day 28. All 18 PRNT80 responders maintained PRNT80 and PRNT50 ≥ 1:40 until at least postvaccination month 12. Viremia was undetectable in the plasma of any subject by direct plaque assay techniques. However, 5 of 19 vaccinees were positive for MP-12 isolates in plasma by blind passage of plasma on Vero cells. Vaccine virus was also recovered from buffy coat material from one of those vaccinees and from one additional vaccinee. Through RNA sequencing of MP-12 isolates, we found no reversions of amino acids to those of the parent virulent virus (strain ZH548). Five years after a single dose of RVF MP-12 vaccine, 8 of 9 vaccinees (89%) maintained a PRNT80 ≥ 1:20. These findings support the continued development of RVF MP-12 as a countermeasure against RVF virus in humans.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyltransferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90–100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine. Published by Elsevier Ltd.

The antibody profile during and after the six-dose primary vaccination series with anthrax vaccine adsorbed (AVA, Biothrax) was characterized in 86 human volunteers. Ninety-three percent of recipients developed IgG antibodies to Bacillus anthracis protective antigen (PA) after two doses, and 100% were seropositive after dose #3. Geometric mean concentrations (GMC) of IgG to PA measured before and after each dose were significantly lower after injection #3 (peak GMC=146.65 microg/mL, trough GMC=15.16 microg/mL) than after injections #4 (peak GMC=430.46 microg/mL, trough GMC=94.57 microg/mL), #5 (peak GMC=415.05 microg/mL, trough GMC=81.94 microg/mL), or #6 (peak GMC=401.16 microg/mL, trough GMC=96.19 microg/mL) (por=0.0001 for each); but not between injections #4 and #5, #5 and #6, or #4 and #6 (por=0.7923 for each). Decay rates for IgG to PA were significantly faster after injection #3 (half life [T1/2]=39.21 days) than after injections #4 (T1/2=72.03 days), #5 (T1/2=70.14 days), and #6 (T1/2=74.59 days) (por=0.0282 for each). Toxin neutralizing assay (TNA) antibody patterns generally paralleled those for IgG to PA. The 6-month dose in the AVA primary series appears to be critical in sustaining IgG to PA concentrations in a substantial proportion of recipients.

Between 1954 and 1973, more than 2000 men entering military service as conscientious objectors participated in Project Whitecoat as medical research volunteers for the Army's biological warfare defense program. An assessment of self-reported, current health status among 358 "exposed" individuals and 164 unexposed control subjects found no conclusive evidence that receipt of investigational agents was related to adverse health outcomes. No differences in current overall health, current exercise levels, self-reported symptoms, and self-reported medical conditions were seen between the study groups. Possible associations were seen between exposure to antibiotics or other biological agents and self-reported asthma (13.0% vs. 2.4%, relative risk [RR] = 6.00, 95% confidence interval [CI] = 1.03-34.90, p = 0.050), as well as between receipt of tularemia vaccine(s) and self-reported asthma (13.3% vs. 2.4%, RR = 6.15, 95% CI = 1.03-36.70, p = 0.049) and increased frequency/severity of headaches (35.6% vs. 18.3%, RR = 2.46, 95% CI = 0.99-6.15, p = 0.074). However, the size of the population under study was insufficient to assert with confidence that these statistical associations are real.

The potency assay currently used to evaluate consistency of manufacture for the anthrax vaccine is contingent upon meeting specified parameters after statistical analysis of the percent survival and time to death of vaccinated guinea pigs after challenge with spores of a virulent strain of Bacillus anthracis. During the development of a new anthrax vaccine based upon recombinant protective antigen (rPA) adsorbed to aluminum hydroxide gel (Alhydrogel), we found that the serological response of female A/J mice, as measured by a quantitative anti-rPA IgG ELISA, may be an effective method to monitor a manufacturer’s consistency for rPA-based vaccines. An advantage of the proposed in vitro-based potency assay is that it will not need stringent biosafety containment measures as required by the current guinea pig potency assay.

A validation of the performance characteristics of a toxin neutralization assay is presented. This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity. This colormetric assay is based upon the reduction of MTT by living cells. Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay. Results showed a high level of repeatability and reproducibility, particularly for a bio-assay. Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio [neutralization ratio (NR)] of the test serum 50% effective dose (ED(50)) to the reference standard ED(50). Accuracy was maintained, even in samples with minimal neutralizing capacity, and linearity was noted when sample dilutions resulted in accurate prediction of the Y(max)and Y(min). Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin. The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin. The assay also remained stable against media and solution storage effects. Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values. The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58).

The pathogenesis of infection by Bacillus anthracis has been the subject of many investigations, but remains incompletely understood. It has been shown that B. anthracis spores germinate in macrophages and perhaps require this intracellular niche to germinate in vivo before outgrowth of the vegetative organism. However, it has also been reported that macrophages are sporicidal in vitro. In our in vivo model, macrophages were depleted from mice by either silica treatment or treatment with liposome-encapsulated dichloromethylene disphosphonate (Cl(2)MDP), and the animals were infected parenterally with virulent ungerminated B. anthracis (Ames strain) spores. The mice in which macrophages had been depleted were killed more rapidly than untreated mice. In addition, augmenting peritoneal populations of macrophages with cultured RAW264.7 cells partially protected mice from disease, increasing the survival rate in a dose dependent relationship. Alveolar macrophages were depleted by intranasal instillation of liposome-encapsulated Cl(2)MDP. The animals with normal alveolar macrophage numbers had significantly greater survival rates after inhaling B. anthracis spores than the macrophage-depleted mice. These findings do not preclude the observations that macrophages provide a site permissive for spore germination, however, these data suggest that macrophages do play an important role in limiting and/or clearing a B. anthracis infection.

In these studies, a serological correlate of protection against anthrax was identified in New Zealand white (NZW) rabbits that had been given one or two injections of various amounts of recombinant protective antigen (rPA) combined with aluminum hydroxide adjuvant (Alhydrogel). Rabbits were subsequently challenged by the aerosol route with spores of the Ames isolate of Bacillus anthracis. Results suggested that the antibody response, as determined by the quantitative anti-rPA IgG ELISA and toxin neutralizing antibody (TNA) assay, were significant predictors (P

In 1999 and 2000 we evaluated a lethal ovitrap (LO) for the control of Aedes aegypti (L.) in three villages in Ratchaburi Province, Thailand. Two blocks of 50 houses (a minimum of 250 m apart) served as treatment and control sites in each village, with each house in the treatment area receiving 10 LOs. Thirty houses in the center of each treatment and control block were selected as sampling sites, with larval and adult mosquito sampling initiated when LOs were placed. Sampling was conducted weekly in 10 of the 30 houses at each site, with each block of 10 houses sampled every third week. Sampling continued for 30 wk. Efficacy of the LO was evaluated by determining number of containers with larvae and/or pupae per house and number of adult mosquitoes collected inside each house. In 1999, the LO had a negligible impact on all measures of Ae. aegypti abundance that were assessed; however, fungal contamination of insecticide-impregnated strips may have been responsible for the low efficacy. In 2000, significant suppression was achieved based on changes in multiple entomologic criteria (containers with larvae, containers with pupae, and number of adult Ae. aegypti); however, control was not absolute and neither immature nor adult Ae. aegypti were ever eliminated completely. We conclude that the LO can reduce adult Ae. aegypti populations in Thailand; however, efficacy of the LO is lower than desired due primarily to the high number of alternative oviposition sites. LO efficacy may be improved when used as part of an integrated control program that places emphasis on reduction of adjacent larval habitats. Further studies are required to assess this issue.

Tularemia is a zoonotic disease caused by Francisella tularensis, which is transmitted to humans most commonly by contact with infected animals, tick bites, or inhalation of aerosolized bacteria. F. tularensis is highly infectious via the aerosol route; inhalation of as few as 10-50 organisms can cause pneumonic tularemia. Left untreated, the pneumonic form has more than > 30% case-fatality rate but with early antibiotic intervention can be reduced to 3%. This study compared tularemia disease progression across three species of nonhuman primates [African green monkey (AGM), cynomolgus macaque (CM), and rhesus macaque (RM)] following aerosolized F. tularensis Schu S4 exposure. Groups of the animals exposed to various challenge doses were observed for clinical signs of infection and blood samples were analyzed to characterize the disease pathogenesis. Whereas the AGMs and CMs succumbed to disease following challenge doses of 40 and 32 colony forming units (CFU), respectively, the RM lethal dose was 276,667 CFU. Following all challenge doses that caused disease, the NHPs experienced weight loss, bacteremia, fever as early as 4 days post exposure, and tissue burden. Necrotizing-to-pyogranulomatous lesions were observed most commonly in the lung, lymph nodes, spleen, and bone marrow. Overall, the CM model consistently manifested pathological responses similar to those resulting from inhalation of F. tularensis in humans and thereby most closely emulates human tularemia disease. The RM model displayed a higher tolerance to infection and survived exposures of up to 15,593 CFU of aerosolized F. tularensis.

Defined candidate human vaccines for treating infection by Yersinia pestis , the agent of plague, have been developed. To facilitate evaluation of the vaccines' efficacy, the in vitro caspase-3 assay for cytotoxicity-neutralizing activity was modified and reevaluated. Immune serum-mediated caspase-3 neutralizing activity correlated with protection against infection in a nonhuman primate vaccine model of plague immunity.

Pneumonic plague is a severe, rapidly progressing disease for which there is no effective vaccine. Since the efficacy of new vaccines cannot be tested in humans, it is essential to develop in vitro surrogate assays that are valid predictors of immunity. The F1 capsule antigen stimulates a protective immune response to most strains ofYersinia pestis. However, strains ofY. pestisthat are F1−but still virulent have been isolated, and an in vitro assay, the results which can predict protection against both F1+and F1−strains, is needed. The virulence antigen (V) is an essential virulence factor ofY. pestisand stimulates protective antibodies. We investigated potential correlates of plague immunity that are based on anti-V antibody-mediated neutralization ofYersinia-induced macrophage cytotoxicity. The neutralizing activity of sera from mice vaccinated with an F1-V fusion candidate vaccine was determined. The decrease in the level of the apoptosis-specific enzyme caspase-3 significantly predicted survival in one- and two-dose vaccination experiments. Sera from F1-V-vaccinated nonhuman primates were evaluated with macrophage assays based on caspase-3 and on other markers manifested at the different stages in cell death. Using murine- and human-derived macrophages in microscopic and fluorescence-activated-cell-sorting-based live/dead staining assays of terminal necrosis, we demonstrated a strong association between in vitro neutralization of macrophage cytotoxicity induced by serum-treatedYersiniaand in vivo protection against lethal infection. These results provide a strong base for the development of reliable in vitro correlate bioassays that are predictive of protective immunity to plague.

We examined, by enzyme-linked immunosorbent assay and Western blot analysis, the host immune response to 2 heat-shock proteins (hsps) in a patient and mice previously infected with Burkholderia mallei. The patient was the first reported human glanders case in 50 years in the United States. The expression of the groEL and dnaK operons appeared to be dependent upon a σ 32 RNA polymerase as suggested by conserved heat-shock promoter sequences, and the groESL operon may be negatively regulated by a controlling invert repeat of chaperone expression (CIRCE) site. In the antisera, the GroEL protein was found to be more immunoreactive than the DnaK protein in both a human patient and mice previously infected with B. mallei. Examination of the supernatant of a growing culture of B. mallei showed that more GroEL protein than DnaK protein was released from the cell. This may occur similarly within an infected host causing an elevated host immune response to the B. mallei hsps. Published by Elsevier Inc.

The serological response and efficacy of Bacillus anthracis recombinant protective antigen (rPA) vaccines formulated with aluminum hydroxide adjuvant, either with or without formaldehyde, were evaluated in rabbits. Rabbits that had been injected with a single dose of 25 microg of rPA adsorbed to 500 microg of aluminum in aluminum hydroxide gel (Alhydrogel) had a significantly higher quantitative anti-rPA IgG ELISA titers (p

A retrospective review was conducted of yellow fever vaccination among laboratory workers receiving annual serologic assessment to determine the initial and long-term response after boosting. Patients were divided into three groups based on pre-vaccination serology: Group 1, 1:10; Group 2, 1:20–1:40 and Group 3, >1:40. The percent with ≥four-fold increase in titers after booster vaccination were: 78% (646/829, Group 1), 65% (79/121, Group 2) and 10% (8/79, Group 3) (p < 0.0001). The median times to titer failure (

Long-term protection of rabbits that had been vaccinated with two doses of a recombinant protective antigen (rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival was 74.1% (20/27) with quantitative ELISA titer of 22.3g of anti-rPA IgG per millilitre and toxin neutralizing antibody (TNA) assay titer of 332. At 12 months after the primary injection, only 37.5% (9/24) of the rabbits were protected with quantitative ELISA titer of 19.8 g of anti-rPA IgG per millilitre and TNA assay titer of 286. There was a significant loss of protection ( p = 0.0117) and a significant difference in survival curves (p = 0.0157) between the 6- and 12-month groups. When ELISA or TNA assay titer, gender, and challenge dose were entered into a forward logistic regression model, week 26 ELISA titer (p = 0.0236) and week 13 TNA assay titer (p = 0.0147) for the 6-month group, and week 26 ELISA titer (p = 0.0326) and week 8 TNA assay titer (p = 0.0190) for the 12-month group, were significant predictors of survival. Neither gender nor challenge dose were identified as having a statistically significant effect on survival. Booster vaccinations with rPA may be required for the long-term protection of rabbits against anthrax. Published by Elsevier Ltd.

Recipients of licensed anthrax vaccine (AVA; Biothrax) could serve as a source of hyperimmune plasma and immunoglobulin for therapy and prophylaxis. We measured serum antibodies during serial weekly to biweekly plasmapheresis in 38 individuals previously vaccinated with 4 to 27 doses of AVA. Immunoglobulin G (IgG) to protective antigen (PA) and toxin neutralization assay (TNA) antibody levels were highly correlated ( r = 0.86930 and P < 0.0001 for anti-PA concentration versus TNA concentration). Significant decreases in antibody titer and concentration were observed over time when compared for the number of days from the last AVA injection ( P < 0.0001 for both anti-PA and TNA concentration) and for the number of days from the first plasmapheresis ( P = 0.0007 for anti-PA concentration and P = 0.0025 for TNA concentration). The rate of the decrease in total IgG concentration (half-life [ t 1/2 ] = 198.90 days after first plasmapheresis) was significantly less than the decrease in anti-PA IgG ( t 1/2 = 63.53 days) ( P < 0.0001), indicating that the reduction in anti-PA IgG was more likely due to natural decay than plasmapheresis. The time since the last injection and the time after initial plasmapheresis are important elements in considering an optimal schedule for collecting anthrax hyperimmune plasma. Good correlation between IgG to PA and TNA antibodies suggests that the anti-PA enzyme-linked immunosorbent assay can be used as a high-throughput screen for functional immune reactivity in donor plasma units.

We evaluated the effect of interleukin (IL)-12 on the immune response to Burkholderia mallei in BALB/c mice. Mice were vaccinated with non-viable B. mallei cells with or without IL-12. There was a seven- to nine-fold increase in IgG2a levels, and a significant increase in the proliferative response and interferon (IFN)- production by splenocytes from mice that received B. mallei and IL-12. We saw an increase in survivors in the groups of mice that received B. mallei and IL-12 when challenged, compared to mice that received only B. mallei or IL-12. The results suggest that IL-12 can enhance the Th1-like immune response to B. mallei and mediate limited protection from a lethal challenge. © 2005 Elsevier Ltd. All rights reserved.

Efficacy of a poly-γ- d -glutamic acid anthrax capsule vaccine was assessed in a mouse model of infection. Capsule by itself was protective against lethal challenge with a toxin − , capsule + Bacillus anthracis strain. Conjugation of capsule to bovine serum albumin resulted in enhanced IgG anti-capsule antibodies measured by ELISA, but completely abrogated the protection. The protective unconjugated capsule vaccine elicited significantly higher IgM titers and opsonic activity than did the non-protective capsule conjugate. When tested against a fully virulent toxin + , capsule + B. anthracis strain, neither capsule nor protective antigen alone was protective. However, the combination of the two protected against a lethal challenge. These results suggest that capsule may enhance the protection afforded by protective antigen vaccines against anthrax if opsonizing antibodies are produced. Surprisingly, some protection was also observed when protective antigen was conjugated to itself.

Mice and guinea pigs were experimentally exposed to aerosols containing regionally-distinct strains (NJ1959 or ArgM) of eastern equine encephalitis virus (EEEV) at two exclusive particle size distributions. Mice were more susceptible to either strain of aerosolized EEEV than were guinea pigs; however, clinical signs indicating encephalitis were more readily observed in the guinea pigs. Lower lethality was observed in both species when EEEV was presented at the larger aerosol distribution (> 6 μm), although the differences in the median lethal dose (LD50) were not significant. Virus isolation and immunohistochemistry indicated that virus invaded the brains of guinea pigs within one day postexposure, regardless of viral strain or particle size distribution. Immunohistochemistry further demonstrated that neuroinvasion occurred through the olfactory system, followed by transneuronal spread to all regions of the brain. Olfactory bipolar neurons and neurons throughout the brain were the key viral targets. The main microscopic lesions in infected guinea pigs were neuronal necrosis, inflammation of the meninges and neuropil of the brain, and vasculitis in the brain. These results indicate that guinea pigs experimentally infected by aerosolized EEEV recapitulate several key features of fatal human infection and thus should serve as a suitable animal model for aerosol exposure to EEEV.