Rapid Establishment of a Biospecimen Resource To Study the Global Impact of COVID-19 Vaccines.

The emergence and explosive spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 highlighted the need to rapidly develop curated biobanks to inform the etiology, diagnosis, and treatment options for global outbreaks of communicable diseases. Recently, we undertook efforts to develop a repository of biospecimens from individuals aged 12 and older who were to be vaccinated against coronavirus disease 19 (COVID-19) with vaccines developed with support from the United States Government. We planned to establish 40 or more clinical study sites in at least six countries to collect biospecimens from 1,000 individuals, 75% of whom were to be SARS-CoV-2 naive at the time of enrollment. Specimens would be used to (i) ensure quality control of future diagnostic tests, (ii) understand immune responses to multiple COVID-19 vaccines, and (iii) provide reference reagents for the development of new drugs, biologics, and vaccines. Biospecimens included serum, plasma, whole blood, and nasal secretions. Large-volume collections of peripheral blood mononuclear cells (PBMCs) and defibrinated plasma were also planned for a subset of subjects. Participant sampling was planned at intervals prior to and following vaccination over a 1-year period. Here, we describe the selection of clinical sites for specimen collection and processing, standard operating procedure (SOP) development, design of a training program for tracking specimen quality, and specimen transport to a repository for interim storage. This approach allowed us to enroll our first participants within 21 weeks from the study's initiation. Lessons learned from this experience should benefit the development of biobanks in response to future global epidemics. IMPORTANCE The ability to rapidly create a biobank of high-quality specimens in response to emergent infectious diseases is critical to allow for the development of prevention and treatment, as well as to effectively monitor the spread of the disease. In this paper, we report on a novel approach to getting global clinical sites up and running within a short time frame and to monitor the quality of specimens collected to ensure their value in future research efforts. Our results have important implications for the monitoring of the quality of biospecimens collected and to design effective interventions to address shortcomings, where needed.

Deficiency of suppressor T cells in the hyperimmunoglobulin E syndrome.

The status of suppressor T cells (Ts) was assessed in seven children with the hyper IgE syndrome (recurrent staphylococcal infections, eczematous skin rash, and elevated serum IgE) to determine whether a deficiency in Ts is associated with increased IgE synthesis. When circulating T cells and their subsets were enumerated with the aid of monoclonal antibodies that identify T cells (T3), helper/inducer T cells (T4), and suppressor/cytotoxic T cells (T8), there was a selective deficiency of T3+ cells (51.7+/-11.2% vs. 66+/-5% for normal controls) and of T8+ cells (7.5+/-4.4% vs. 22+/-4% for normal controls) but not of T4+ cells (36.5+/-7.5% vs. 37+/-3% for normal controls). Suppressor T cell function was assessed by examining the ability of mononuclear cells incubated for 48 h with concanavalin A to suppress the proliferation of fresh autologous mononuclear cells in response to the mitogens phytohemagglutinin and pokeweed mitogen. All seven patients were severely deficient in concanavalin A-inducible suppressor cells. In vitro de novo synthesis of IgE in 6-d cultures of peripheral blood lymphocytes was measured in four patients by a solid-phase radioimmunoassay. Mononuclear cells from all four patients synthesized spontaneously increased quantities of IgE in vitro (4,950+/-3,760 pg/10(6) cells vs. 250+/-215 pg/10(6) cells for eight normal controls). IgE synthesis was suppressed by the addition of parental T cells to the culture. Elimination of the T8+ subset, but not of the T4+ subset, by complement-dependent lysis resulted in the loss of the capacity of parental T cells to suppress IgE synthesis. These results suggest that a deficiency of Ts underlies the elevated IgE levels observed in the hyper IgE syndrome.

Cloning and expression of three isoforms of the human EP3 prostanoid receptor.

Functional cDNA clones coding for three isoforms of the human prostaglandin E receptor EP3 subtype have been isolated from kidney and uterus cDNA libraries. The three isoforms, designated hEP3-I, hEP3-II and hEP3-III, have open reading frames corresponding to 390, 388 and 365 amino acids, respectively. They differ only in the length and amino acid composition of their carboxy-terminal regions, beginning at position 360. The human EP3 receptor has seven predicted transmembrane spanning domains and therefore belongs to the G-protein-coupled receptor family. The rank order of potency for prostaglandins and related analogs in competition for [3H]PGE2 specific binding to membranes prepared from transfected COS cells was comparable for all three isoforms, and as predicted for the EP3 receptor, with PGE2 = PGE1 >> PGF2 alpha = iloprost > PGD2 >> U46619. In addition, the EP3-selective agonist MB28767 was a potent competing ligand with an IC50 value of 0.3 nM, whereas the EP1-selective antagonist AH6909 gave IC50 values of 2-7 microM and the EP2-selective agonist butaprost was inactive. In summary, we have cloned three isoforms of the human EP3 receptor having comparable ligand binding properties.

Patterns of illness in the highly febrile young child: epidemiologic, clinical, and laboratory correlates.

Three hundred one episodes of fever greater than or equal to 103 F were documented in 375 infants and young children observed in a comprehensive care clinic during the period October 1974 to October 1978. Of such highly febrile illnesses 79% were accompanied by respiratory tract signs or symptoms, 7% by disease at a site other than the respiratory tract, and 22% of illnesses had no localizing signs or symptoms. Viral cultures were obtained from the respiratory tract in 178 cases and were positive in 68: 57/134 from respiratory illness; 2/4 from illness at sites other than the respiratory tract; and 9/40 in children without localizing disease. Bacterial cultures of the upper respiratory tract were obtained in 191 illnesses, but the overall rate of isolation of Haemophilus influenzae, Streptococcus pneumoniae and group A streptococci (46%) did not differ from that in a group of well children (39%). Bacterial cultures of the blood were obtained in 89 patients with fever greater than or equal to 103 F and in an additional 41 children with lower temperatures. Nine children had documented systemic bacterial disease (eight positive blood cultures and one positive CSF). The rate of clinically apparent systemic bacterial disease in these otherwise normal infants was one bacteremic episode per 94 years of child care.

Identification of both NK1 and NK2 receptors in guinea-pig airways.

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P-methyl ester = physalaemin > neurokinin A = neurokinin B >> [beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations. 5. Similar studies using guinea-pig tracheal membranes demonstrated that [125I]-neurokinin A specific binding was composed of a NK1-receptor component (60%), inhibited by both [Sar9Met(02)11]substance P and CP-99,994, and a significant NK2-receptor component, inhibited by both [beta Ala 8]neurokinin A andSR48968.6. In summary, these data demonstrate that guinea-pig lung parenchyma and guinea-pig trachea express both NK1 and NK2 receptors.

Pathogenesis of Hantaan virus infection in suckling mice: clinical, virologic, and serologic observations.

Hemorrhagic fever with renal syndrome (HFRS) is a debilitating disease of humans caused by Hantaan virus (HV), the prototype member of a newly proposed genus of Bunyaviridae. Studies of HV pathogenesis have been limited by the absence of a well defined model for a virus-induced disease state. In an attempt to devise a model for HV pathogenesis in laboratory rodents, newborn outbred suckling ICR mice were shown to be uniformly susceptible to lethal infection with non-mouse adapted HV by intracerebral (IC), intraperitoneal (IP), intramuscular (IM), and subcutaneous (SC) inoculation routes. Clinical course, mean time to death, and fatal outcome were age-dependent. With an inoculum of 10 LD50, mortality was 100% in mice infected within 72 hr of birth, but declined to 50% by 7 days. By 2-2.5 weeks, animals developed complete resistance to clinical disease. Virus was consistently detected in serum by day 6 post-infection in IC- and IP-inoculated animals, and reached peak levels of congruent to 10(5) PFU/ml by day 8. Mice infected IM and SC showed delays in onset of viremia, but achieved similar titers. Immunofluorescent antibody appeared by 17-18 days, and neutralizing antibody by 15 days, in all experimental groups. Two of 8 inbred mouse strains were identified as resistant to clinical disease: SJL/J and A/J.

Outbreak of hemorrhagic fever with renal syndrome among U.S. Marines in Korea.

Fourteen of 3,754 U.S. Marines who participated in a joint United States-Republic of Korea training exercise during the autumn of 1986 developed hemorrhagic fever with renal syndrome (HFRS). Clinical and laboratory findings among cases included fever, headache, fatigue, gastrointestinal dysfunction, thrombocytopenia, and proteinuria. Ten individuals were hospitalized; 2 died. No subclinical infections were identified through a post-deployment screen of sera obtained from 2,053 exercise participants. Analysis of questionnaires identified no environmental, occupational, or temporal factors as risks for developing disease. However, 13 of the 14 cases occurred among individuals housed at 1 of the 2 base camps used during the exercise. This outbreak represents the largest cluster of HFRS cases among U.S. personnel in the Republic of Korea since the Korean conflict.

Safety and immunogenicity of a live-attenuated Junin (Argentine hemorrhagic fever) vaccine in rhesus macaques.

The safety and immunogenicity of Candid #1, a live-attenuated Junin virus vaccine, were evaluated in rhesus macaques. Candid #1 was inoculated subcutaneously in graded doses ranging from 16 to 127,200 plaque-forming units (PFU) into four groups of five animals each; four controls received saline. There was no significant effect of the immunization on any physical, hematologic, or biochemical parameter measured. Junin virus was recovered by cocultivation from peripheral blood mononuclear cells (PBMC) of 14 (70%) of 20 animals from 1 to 21 days after immunization; 27 (12%) of 223 PBMC samples that represented animals in all four dose groups were positive. In contrast, virus was recovered from the plasma of only two of 20 macaques (two of 225 samples [0.9%]), and only once (by amplification) from throat swabs. No evidence of reversion was detected in any blood isolate. All animals developed a detectable neutralizing antibody response following vaccination. These results indicate that Candid #1 is safe and immunogenic in nonhuman primates.

Experimental infection of Phlebotomus papatasi with sand fly fever Sicilian virus.

Experimental studies were conducted to evaluate humans as hosts infecting the sand fly Phlebotomus papatasi with sand fly fever Sicilian (SFS) virus. Viral antigen and infectious virus circulated in the blood of infected volunteers on days 4 and 5 after intravenous inoculation with SFS virus. Viremia levels during the latter period were high enough to infect feeding sand flies, but only 13% (9/69) of the flies became infected. One out of every 3 infected sand flies that survived to feed a second time transmitted SFS to a hamster. These results confirm a vertebrate-sand fly-vertebrate transmission cycle for SFS virus, and demonstrate that horizontal transmission may contribute to the maintenance of this virus in nature.

Prevalence of infection with Junin virus in rodent populations in the epidemic area of Argentine hemorrhagic fever.

We report the results of indirect fluorescent antibody screening for antibody to Junin virus in 1,101 sera from small mammals captured on two mark-recapture grids in the epidemic area of Argentine hemorrhagic fever. Twenty-six of 29 seropositive animals were the cricetid rodent Calomys musculinus, for a 30-month prevalence of 7.9% in that species. Combining these data with previously published data on antigen detection provided an estimated total prevalence of infection of 10.9% for this, the principal reservoir species. Other infected species included two cricetids, C. laucha and Bolomys obscurus, and a predatory carnivore, Galictis cuja. Approximately half of infected animals simultaneously carried serum antibody and antigen in blood and saliva, some for 29-61 days. Except for C. laucha, which was associated with crop habitats, seropositive animals were strongly associated with the relatively rare roadside and fence-line habitats. Seropositive C. musculinus were predominantly males in the oldest age and heaviest body mass classes, and seropositive males were twice as likely to have body scars as seronegative males. These observations suggest that most infections were acquired through horizontal transmission and that aggressive encounters among adult, male C. musculinus in relatively densely populated roadside and fence-line habitats are an important mechanism of transmission of Junin virus within reservoir populations.

Lack of attenuation of a candidate dengue 1 vaccine (45AZ5) in human volunteers.

A dengue type 1, candidate live virus vaccine (45AZ5) was prepared by serial virus passage in fetal rhesus lung cells. Infected cells were treated with a mutagen, 5-azacytidine, to increase the likelihood of producing attenuated variants. The vaccine strain was selected by cloning virus that produced only small plaques in vitro and showed reduced replication at high temperatures (temperature sensitivity). Although other candidate live dengue virus vaccines selected for similar growth characteristics have been attenuated for humans, two recipients of the 45AZ5 virus developed unmodified acute dengue fever. Viremia was observed within 24 hr of inoculation and lasted 12 to 19 days. Virus isolates from the blood produced large plaques in cell culture and showed diminished temperature sensitivity. The 45AZ5 virus is unacceptable as a vaccine candidate. This experience points out the uncertain relationship between in vitro viral growth characteristics and virulence factors for humans.

A longitudinal study of Junin virus activity in the rodent reservoir of Argentine hemorrhagic fever.

We monitored Junin virus (JV) activity in rodent populations for 30 months at seven mark-recapture grids located in agricultural fields and adjacent roadsides and fence lines in endemic and nonendemic areas of Argentine hemorrhagic fever. Blood and oral swabs taken from rodents captured at five-week intervals were analyzed by enzyme-linked immunosorbent assay for JV antigen (Ag). Calomys laucha and C. musculinus were the most frequently captured rodents, making up 47% and 22% of captures, respectively. Of 41 Ag-positive captures, 37 were C. musculinus and four were C. laucha; 34 were from two trapping grids in the same locality. Antigen-positive Calomys were more frequently male (76%), and were found significantly more frequently among the oldest animals and the largest body mass classes. These patterns, combined with the greater mobility and higher frequencies of wounds among males than females, implicated horizontal transmission as the primary route of JV transmission between rodents. Seasonal maximum levels in JV prevalence (up to 25% of captured Ag-positive C. musculinus) occurred during periods of maximal population densities of Calomys. Spatial distribution of Ag-positive rodents reflected habitat preferences; most Ag-positive C. musculinus were captured from border habitats (roadsides and fence lines), and all Ag-positive C. laucha were captured in crop fields. These distinct, but previously undocumented, habitat preferences suggest that the disease in humans may be related to exposures to the primary reservoir species, C. musculinus, in border habitats rather than in crop fields.

Junin virus activity in rodents from endemic and nonendemic loci in central Argentina.

Small mammals were trapped during a 21-month period at 27 farm sites in 15 localities within and beyond the known endemic area for Argentine hemorrhagic fever (AHF). Prevalence of Junin virus (JV) was assessed by antigen-capture enzyme immunoassay (ELISA) on samples of body fluids and/or organs from 3, 282 captured rodents. Infection in rodent populations was variable (0-3.7%) among localities but, in all cases, was lower than previously reported rates. Overall prevalence was 1.4% in the AHF epidemic area, 0.6% in the historic (currently low incidence of AHF) area, and 0.4% in two localities beyond the previously defined endemic area. These low values underestimate the actual prevalence of JV, as ELISA validation by virus isolation indicated a sensitivity of 30% and a specificity of 99%. Of 37 positive rodents, 28 (76%) were of two species: Calomys musculinus (23 animals) and C. laucha (5 animals). Antigen also was found in three Akodon azarae, four Bolomys obscurus, one Mus musculus, and one Oxymycterus rufus, and JV was isolated from two Oligoryzomys flavescens. Three of these rodent species (B. obscurus, O. flavescens, and O. rufus) have heretofore not been implicated in JV maintenance in the field. Evidence suggests that the AHF endemic area may continue to expand northward.

Characterization of Oliveros virus, a new member of the Tacaribe complex (Arenaviridae: Arenavirus).

Oliveros virus is an agent isolated in cell culture from Bolomys obscurus (Rodentia, Muridae, Sigmodontinae) captured on the central Argentine pampa. Oliveros virus was shown to be related to members of the Tacaribe complex of the family Arenaviridae by immunofluorescent antibody (IFA) tests, electrophoretic pattern of viral proteins, and morphology as observed by electron microscopy. It was distinct from 12 other arenaviruses by a combination of plaque-reduction neutralization tests, comparison of endpoint titers among cross-IFA tests, and comparison of viral RNA sequence data. This agent is the third new arenavirus from South America described within the last three years.

A refined complement-enhanced neutralization test for detecting antibodies to Junin virus.

A refined, complement-enhanced, plaque-reduction neutralization test was developed for measuring neutralizing antibodies against Junin (Argentine hemorrhagic fever) virus. The assay measured neutralizing antibodies after natural as well as vaccine-induced Junin virus infections. Among vaccinated individuals, titers were 2-4-fold higher than those obtained with conventional assays, without loss of specificity. Enhanced sensitivity was achieved by using a standardized complement source (vs human or animal serum) for virus dilution, incubation of virus-serum mixtures at 36 degrees C for 2 h (vs overnight at 4 degrees C) prior to plaque assay, control of age and density of cell monolayers, and variation in overlay conditions.

Rubella outbreak, Fort Bragg, North Carolina, 1995: a clash of two preventive strategies.

An outbreak of rubella occurred among visiting German troops involved in a combined military exercise at Fort Bragg, North Carolina, in April 1995. Public health and military operational concerns centered on the significant contact the German soldiers had had with host battalion dependents and the impact of the outbreak on the exercise. Ten of the 120 German soldiers were found to be nonimmune; six of these soldiers developed clinical rubella. The four nonimmune soldiers who did not develop skin rashes had received serum immune globulin within 12 hours of identification of the index case. The impact of this outbreak on the Fort Bragg community and its military operations, and the methods used to control the outbreak and salvage the military mission, are described.

Disease and nonbattle injury among United States soldiers deployed in Bosnia-Herzegovina during 1997: summary primary care statistics for Operation Joint Guard.

Systematic surveillance of outpatient (primary care) encounters with the health care system has been performed for North Atlantic Treaty Organization coalition forces during peace-keeping operations in Bosnia-Herzegovina since 1995. The present study presents an analysis of disease and nonbattle injury (DNBI) surveillance findings for U.S. forces participating in Operation Joint Guard during 1997. The mean DNBI rate for this 1-year period was 8.1/100/week (range, 5.7-11.1/100/week). Most frequently cited causes for soldier visits to medical treatment facilities were injuries and orthopedic conditions (27%), respiratory disease (26%), miscellaneous "other" medical conditions (13%), dermatologic disorders (12%), and dental disease (10%). Gastroenteritis was infrequently seen (2% of visits). Our findings extend previous observations that indicate that the Bosnia peacekeeping mission is relatively safe and healthy for U.S. forces.

Early syphilis in an active duty military population and the surrounding civilian community, 1985-1993.

Syphilis among active duty soldiers at Fort Bragg, North Carolina, and the nonmilitary population of Cumberland County was examined during a 9-year period encompassing the most recent nationwide syphilis epidemic. A total of 762 cases of primary and secondary syphilis were recorded between 1985 and 1993, 27% of which occurred in soldiers. The epidemic struck both military and civilian populations simultaneously; epidemic curves in the two populations were parallel, peaking in 1990-1991, with highest annual incidences of 122.6/ 100,000 (military) and 48.0/100,000 (civilian). Individual risk factor data were not available for analysis, but a relationship was observed between primary and secondary syphilis diagnoses in both populations and cocaine arrests in Cumberland County. Our findings provide epidemiological support for a high degree of interplay between the military and the surrounding civilian communities that has significant implications for control of sexually transmitted diseases. Enhanced collaboration between military and civilian public health authorities is essential to the control of syphilis and other sexually transmitted diseases.

Human immunodeficiency virus (HIV) education and HIV risk behavior: a survey of rapid deployment troops.

A paper-and-pencil questionnaire was administered to 1,377 U.S. Army troops from rapid deployment units at Fort Bragg, North Carolina. This yielded 1,368 surveys available for analysis. The primary goal of the survey was to evaluate this group's experience with the Army human immunodeficiency virus (HIV) education program and to determine their level of HIV risk behaviors as related to participation in the Army's HIV education program. Seventy-seven percent of the respondents (1,052 of 1,368) reported receiving some HIV education from the Army. Of those, 55% (578 of 1,052) reported receiving 1 hour of education within the past year. Soldiers of Asian, Native American, and "other" race/ethnicity, and to a lesser extent, Hispanic background, were more likely to report receiving no HIV education compared with whites and African Americans. Self-reported receipt of HIV education did not strongly differentiate individuals in their partner selection or in key sexual risk behaviors in which they engaged.

[New findings on Junin virus infection in rodents inside and outside the endemic area of hemorrhagic fever in Argentina]. / Nuevas observaciones de la infección de roedores por el virus Junin dentro y fuera de la zona endemica de la Fiebre Hemorragica Argentina.

In conjunction with field trials for a vaccine against Argentine Hemorrhagic Fever (AHF), small mammals were trapped during a 28-month period (1 November 1987 to 13 March 1990) in 3 epidemiologically defined areas of the central Argentine pampas: northern and central Buenos Aires provinces were included in the AHF "historic" area, where the disease was common 15-20 years ago, but case rates are currently low; southern Santa Fe province is the current high-incidence area for AHF; the nonendemic area was represented by two localities 60-90 km beyond the northernmost extension of human disease. Animals were live-trapped for 3 days per month in permanent "mark-recapture" grids in each of the 3 areas. Samples of blood, sera, and oral swabs were taken from these animals before they were marked and released at the site of capture. In addition, "removal" traplines provided animals from 16 localities in these 3 areas which were sacrificed to obtain samples of organs in addition to the aforementioned samples. Samples were tested for the presence of Junin virus (JV) antigen by enzyme immunoassay (ELISA). In this assay, a pool of 13 mouse anti-JV glycoprotein and nucleocapsid monoclonal antibodies adsorbed to the surface of microtiter plates was used to capture JV antigen in sample suspensions. A polyclonal rabbit anti-JV antiserum was added as a detector antibody, and an anti-rabbit antibody conjugated to horseradish peroxidase applied with substrate to complete the sandwich.(ABSTRACT TRUNCATED AT 250 WORDS)