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Digital therapeutics is a newly described concept in healthcare which is proposed to change patient behavior and treat medical conditions using a variety of digital technologies. However, the term is rarely defined with criteria that make it distinct from simply digitizedversions of traditional therapeutics. Our objective is to describe a more valuable characteristic of digital therapeutics, which is distinct from traditional medicine or therapy: that is, the utilization of artificial intelligence and machine learning systems to monitor and predict individual patient symptom data in an adaptive clinical feedback loop via digital biomarkers to provide a precision medicine approach to healthcare. Artificial intelligence platforms can learn and predict effective interventions for individuals using a multitude of personal variables to provide a customized and more tailored therapy regimen. Digital therapeutics coupled with artificial intelligence and machine learning also allows more effective clinical observations and management at the population level for various health conditions and cohorts. This vital differentiation of digital therapeutics compared to other forms of therapeutics enables a more personalized form of healthcare that actively adapts to patients’ individual clinical needs, goals, and lifestyles. Importantly, these characteristics are what needs to be emphasized to patients, physicians, and policy makers to advance the entire field of digital healthcare.

FDA-approved and emergency use-authorized vaccines using new mRNA and viral-vector technology are highly effective in preventing moderate to severe disease; however, information on their long-term efficacy and protective breadth against severe acute respiratory syndrome coronavirus 2 variants of concern (VOCs) is currently scarce. Here, we describe the durability and broad-spectrum VOC immunity of a prefusion-stabilized spike (S) protein adjuvanted with liquid or lyophilized CoVaccine HT in cynomolgus macaques. This recombinant subunit vaccine is highly immunogenic and induces robust spike-specific and broadly neutralizing antibody responses effective against circulating VOCs (B.1.351 [Beta], P.1 [Gamma], and B.1.617 [Delta]) for at least three months after the final boost. Protective efficacy and postexposure immunity were evaluated using a heterologous P.1 challenge nearly three months after the last immunization. Our results indicate that while immunization with both high and low S doses shorten and reduce viral loads in the upper and lower respiratory tract, a higher antigen dose is required to provide durable protection against disease as vaccine immunity wanes. Histologically, P.1 infection causes similar COVID-19-like lung pathology as seen with early pandemic isolates. Postchallenge IgG concentrations were restored to peak immunity levels, and vaccine-matched and cross-variant neutralizing antibodies were significantly elevated in immunized macaques indicating an efficient anamnestic response. Only low levels of P.1-specific neutralizing antibodies with limited breadth were observed in control (nonvaccinated but challenged) macaques, suggesting that natural infection may not prevent reinfection by other VOCs. Overall, these results demonstrate that a properly dosed and adjuvanted recombinant subunit vaccine can provide protective immunity against circulating VOCs for at least three months.

Lassa Fever (LF) is an acute viral hemorrhagic fever caused by Lassa virus (LASV) that is primarily transmitted through contact with wild rodents in West Africa. Although several advanced vaccine candidates are progressing through clinical trials, some effective vaccines are virally vectored and thus require a stringent cold-chain, making distribution to rural and resource-poor areas difficult. Recombinant subunit vaccines are advantageous in this aspect as they can be thermostabilized and deployed with minimal storage and transportation requirements. However, antigen dose and adjuvant formulation must be carefully selected to ensure both the appropriate humoral and cell-mediated immune responses are elicited. In this study, we examine the immunogenicity of a two-step immunoaffinity-purified recombinant LASV glycoprotein (GP) with five clinical- and preclinical-grade adjuvants. Swiss Webster mice immunized intramuscularly with 2 or 3 doses of each vaccine formulation showed complete seroconversion and maximal GP-specific antibody response after two immunizations. Formulations with GPI-0100, LiteVax, Montanide™ ISA 51, and Montanide™ ISA 720 induced both IgG1 and IgG2 antibodies suggesting a balanced Th1/Th2 response, whereas formulation of LASV GP with Alhydrogel elicited a IgG1-dominant response. Splenocytes secreting both Th1 and Th2 cytokines i.e., IFN-γ, TNF-α, IL-2, IL-4 and IL-5, were observed from mice receiving both antigen doses formulated with ISA 720, LiteVax and GPI-0100. However, robust, multifunctional T-cells were only detected in mice receiving a higher dose of LASV GP formulated with GPI-0100. Our results emphasize the importance of careful adjuvant selection and lay the immunological basis for a recombinant subunit protein LF vaccine formulation.

Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus are the filoviruses most commonly associated with human disease. Previously, we administered a three-dose regimen of trivalent vaccines comprising glycoprotein antigens from each virus in mice and non-human primates (NHPs). The vaccines, which contained a polysorbate 80-stabilized squalane-in-water emulsion adjuvant and were lyophilized from a solution containing trehalose, produced high antibody levels against all three filovirus antigens. Subsequently, single-vial formulations containing a higher concentration of adjuvant were generated for testing in NHPs, but these vaccines elicited lower neutralizing antibody titers in NHPs than previously tested formulations. In order to explain these results, in the current work we measured the size of adjuvant emulsion droplets and the peroxide levels present in the vaccines after lyophilization and reconstitution and tested the effects of these variables on the immune response in mice. Increases in squalane droplet sizes were observed when the ratio of adjuvant to trehalose was increased beyond a critical value, but antibody and neutralizing antibody titers in mice were independent of the droplet size. Higher levels of peroxides in the vaccines correlated with higher concentrations of adjuvant in the formulations, and higher peroxide levels were associated with increased levels of oxidative damage to glycoprotein antigens. Neutralizing titers in mice were inversely correlated with peroxide levels in the vaccines, but peroxide levels could be reduced by adding free methionine, resulting in retention of high neutralizing antibody titers. Overall, the results suggest that oxidation of glycoprotein antigens by peroxides in the polysorbate 80-stabilized squalane-in-water emulsion adjuvant, but not lyophilization-induced increases in adjuvant emulsion droplet size may have been responsible for the decreased neutralizing titers seen in formulations containing higher amounts of adjuvant.

FDA-approved and Emergency Use Authorized (EUA) vaccines using new mRNA and viral-vector technology are highly effective in preventing moderate to severe disease, however, information on their long-term efficacy and protective breadth against SARS-CoV-2 Variants of Concern (VOCs) is currently scarce. Here we describe the durability and broad-spectrum VOC immunity of a prefusion-stabilized spike (S) protein adjuvanted with liquid or lyophilized CoVaccine HT™ in cynomolgus macaques. This recombinant subunit vaccine is highly immunogenic and induces robust spike-specific and broadly neutralizing antibody responses effective against circulating VOCs (B.1.351 [Beta], P.1 [Gamma], B.1.617 [Delta]) for at least 3 months after the final boost. Protective efficacy and post-exposure immunity were evaluated using a heterologous P.1 challenge nearly 3 months after the last immunization. Our results indicate that while immunization with both high and low S doses shorten and reduce viral loads in the upper and lower respiratory tract, a higher antigen dose is required to provide durable protection against disease as vaccine immunity wanes. Histologically, P.1 infection causes similar COVID-19-like lung pathology as seen with early pandemic isolates. Post-challenge IgG concentrations were restored to peak immunity levels and vaccine-matched and cross-variant neutralizing antibodies were significantly elevated in immunized macaques indicating an efficient anamnestic response. Only low levels of P.1-specific neutralizing antibodies with limited breadth were observed in control (non-vaccinated but challenged) macaques suggesting that natural infection may not prevent reinfection by other VOCs. Overall, these results demonstrate that a properly dosed and adjuvanted recombinant subunit vaccine can provide long-lasting and protective immunity against circulating VOCs.One Sentence SummaryA recombinant subunit protein formulated with CoVaccine HT™ adjuvant induces superior immunity than natural infection and reduces viral load while protecting cynomolgus macaques from COVID-19-like disease caused by late SARS-CoV-2 P.1 (Gamma) challenge.

Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), and Marburg marburgvirus (MARV) are the most prevalent and pathogenic species of filovirus. Previously, we showed that glycoprotein antigens from each virus could be lyophilized to create thermostable monovalent subunit vaccines. However, cross-protection is not expected from the monovalent vaccines and therefore developing a trivalent filovirus vaccine would be desirable. Subunit protein vaccines often require the addition of an adjuvant to sufficiently boost the immunogenicity. Typically, liquid suspensions or emulsions of adjuvants and lyophilized antigens are stored in separate vials to avoid destabilizing interactions and are only mixed immediately before administration. Herein, we describe the development and characterization of monovalent and trivalent filovirus vaccines that are co-lyophilized with a squalane-in-water emulsion adjuvant. We found that the single-vial presentation retained adjuvant particle diameter and zeta potential after lyophilization and reconstitution. Furthermore, the trivalent vaccines elicited high antibody levels against all three antigens in mice and non-human primates. These results advance the prospect of developing a single-vial trivalent filovirus vaccine, which would enable easier distribution and administration of the vaccine to resource-poor areas.

Ebola (EBOV), Marburg (MARV) and Sudan (SUDV) viruses are the three filoviruses which have caused the most fatalities in humans. Transmission from animals into the human population typically causes outbreaks of limited scale in endemic regions. In contrast, the 2013-16 outbreak in several West African countries claimed more than 11,000 lives revealing the true epidemic potential of filoviruses. This is further emphasized by the difficulty seen with controlling the 2018-2020 outbreak of EBOV in the Democratic Republic of Congo (DRC), despite the availability of two emergency use-approved vaccines and several experimental therapeutics targeting EBOV. Moreover, there are currently no vaccine options to protect against the other epidemic filoviruses. Protection of a monovalent EBOV vaccine against other filoviruses has never been demonstrated in primate challenge studies substantiating a significant void in capability should a MARV or SUDV outbreak of similar magnitude occur. Herein we show progress on developing vaccines based on recombinant filovirus glycoproteins (GP) from EBOV, MARV and SUDV produced using the Drosophila S2 platform. The highly purified recombinant subunit vaccines formulated with CoVaccine HT™ adjuvant have not caused any safety concerns (no adverse reactions or clinical chemistry abnormalities) in preclinical testing. Candidate formulations elicit potent immune responses in mice, guinea pigs and non-human primates (NHPs) and consistently produce high antigen-specific IgG titers. Three doses of an EBOV candidate vaccine elicit full protection against lethal EBOV infection in the cynomolgus challenge model while one of four animals infected after only two doses showed delayed onset of Ebola Virus Disease (EVD) and eventually succumbed to infection while the other three animals survived challenge. The monovalent MARV or SUDV vaccine candidates completely protected cynomolgus macaques from infection with lethal doses of MARV or SUDV. It was further demonstrated that combinations of MARV or SUDV with the EBOV vaccine can be formulated yielding bivalent vaccines retaining full efficacy. The recombinant subunit vaccine platform should therefore allow the development of a safe and efficacious multivalent vaccine candidate for protection against Ebola, Marburg and Sudan Virus Disease.

SummaryBackgroundVoice signal analysis is an emerging non-invasive technique to examine health conditions, and is implemented in various real-life applications and devices. The purpose of this study was to evaluate the association of voice signals with blood glucose levels in healthy individuals. The study aimed to investigate the longitudinal stabilities of voice signals and identify voice biomarkers to predict abnormal blood glucose levels.MethodsWe created voice profiles composed of 17,552,688 voice signals from 44 participants and their 1,454 voice recordings. From each voice recording, 12,082 voice-features were extracted. Longitudinal stabilities of voice-features were quantified using linear mixed-effect modelling. Voice-features that showed significant difference between different blood glucose levels, strong intra-stability and the ability to make distinct choice in decision trees were selected as voice biomarker. Voice biomarkers were fed into a multi-class random forest classifier to predict high, normal, and low blood glucose levels.FindingsIn total, 196 voice biomarkers were characterized. Results showed a predictive model with an overall accuracy of 78.66%, overall AUC of 0.83 (95% confidence interval is 0.80 – 0.85), and 0.41 of Matthews Correlation Coefficient (MCC) to discriminate three different blood glucose levels in an independent test set.InterpretationOur voice biomarkers could serve as a noninvasive and conventional surrogate of blood glucose monitoring in daily life as well as a screening tool to estimate potential risk of poor glycemic control.FundingThis research was internally funded and received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Ebola virus (EBOV) is among the deadliest pathogens known to man causing infrequent outbreaks of hemorrhagic disease. In humans, the case fatality rates in the outbreaks can reach 90%. During the West African epidemic almost 30,000 people were infected and of these over 11,000 fatalities were reported. Currently, we are facing an uncontained larger outbreak in the Democratic Republic of the Congo. Even though EBOV was discovered in 1976, extensive efforts to develop countermeasures, particularly therapeutics and vaccines, started late and there is still no FDA-approved product available. Nevertheless, one candidate vaccine, the rVSV-ZEBOV, is being used in clinical trials during the current outbreak with the hope of ending the human transmission chains. However, adverse reactions to administration of some EBOV vaccines have been reported; therefore, we have developed a safe and efficacious formulation of insect-cell derived adjuvanted protein vaccines. Vaccine candidates containing the EBOV glycoprotein with or without matrix proteins VP24 and VP40 formulated with one of three different adjuvants were tested in guinea pigs for immunogenicity and efficacy against lethal EBOV challenge. The results demonstrated that these vaccine candidates engendered high titers of antigen-specific antibodies in immunized animals and two of these vaccine candidates afforded complete or nearly complete protection against lethal challenge. Interestingly, we found a sex bias in partially protected immunized groups with male guinea pigs succumbing to disease and females surviving. In summary, we developed a safe and immunogenic adjuvanted subunit vaccine uniformly protective against EBOV disease in guinea pigs.

Zika Virus (ZIKV), a virus with no severe clinical symptoms or sequelae previously associated with human infection, became a public health threat following an epidemic in French Polynesia 2013–2014 that resulted in neurological complications associated with infection. Although no treatment currently exists, several vaccines using different platforms are in clinical development. These include nucleic acid vaccines based on the prM-E protein from the virus and purified formalin-inactivated ZIKV vaccines (ZPIV) which are in Phase 1/2 clinical trials. Using a recombinant subunit platform consisting of antigens produced in Drosophila melanogaster S2 cells, we have previously shown seroconversion and protection against viremia in an immunocompetent mouse model. Here we demonstrate the efficacy of our recombinant subunits in a non-human primate (NHP) viremia model. High neutralizing antibody titers were seen in all protected macaques and passive transfer demonstrated that plasma from these NHPs was sufficient to protect against viremia in mice subsequently infected with ZIKV. Taken together our data demonstrate the immunogenicity and protective efficacy of the recombinant subunit vaccine candidate in NHPs as well as highlight the importance of neutralizing antibodies in protection against ZIKV infection and their potential implication as a correlate of protection.

Infections with filoviruses in humans are highly virulent, causing hemorrhagic fevers which result in up to 90% mortality. In addition to natural infections, the ability to use these viruses as bioterrorist weapons is of significant concern. Currently, there are no licensed vaccines or therapeutics available to combat these infections. The pathogenesis of disease involves the dysregulation of the host’s immune system, which results in impairment of the innate and adaptive immune responses, with subsequent development of lymphopenia, thrombocytopenia, hemorrhage, and death. Questions remain with regard to the few survivors of infection, who manage to mount an effective adaptive immune response. These questions concern the humoral and cellular components of this response, and whether such a response can be elicited by an appropriate prophylactic vaccine. The data reported herein describe the production and evaluation of a recombinant subunit Ebola virus vaccine candidate consisting of insect cell expressed Zaire ebolavirus (EBOV) surface glycoprotein (GP) and the matrix proteins VP24 and VP40. The recombinant subunit proteins are shown to be highly immunogenic in mice, yielding both humoral and cellular responses, as well as highly efficacious, providing up to 100% protection against a lethal challenge with live virus. These results demonstrate proof of concept for such a recombinant non-replicating vaccine candidate in the mouse model of EBOV which helps to elucidate immune correlates of protection and warrants further development.

The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques ( Macaca mulatta ). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 μg of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzyme-linked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 μg of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or 25-μg dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.

As we seek to identify new norms to bridge the gaps between extant law and the challenges that new conflict modes pose today, we confront a threshold question as to which methodological ground we should stand upon in doing so. Based on a background assumption of positivism as the source of substantive norms, the issue for some observers comes down to a clash between pragmatism and formalism. To formalism's proponents, the concept of pragmatism—which sees law as a functional instrument to be used in pursuit of pre-envisioned ends—has contributed to a dearth of moral obligation in international humanitarian law discourse. Such a view considers that the emphasis on empiricism found in pragmatism both legitimizes and shrouds the reality of effective power lurking behind the law. The alternative they prefer, championed most articulately by Professor Koskenniemi, is a “culture of formalism” that sees law as an object of universal obligation and as a form of critique that unmasks the pragmatic mode for what it is, namely, a rationalization of might. As this Article suggests, this understanding misapprehends the true nature of pragmatism, which is neither a smokescreen nor an apoloay. Rather, pragmatism's focus on real-world effects and consequences holds far greater promise for advancing the actual humanitarianism of IHL. Formalism, moreover; is subsumed within the constellation of factors that pragmatic analysis demands. These ideas are explored on a theoretical level, and are then illustrated by a look at the Israel separation barrier cases decided by the International Court of Justice and the Israeli High Court of Justice.

Introduction of the West Nile virus (WNV) to Hawai’i will undoubtedly devastate many populations of critically endangered avian species indigenous to Hawai’i. The protective efficacy of a protein-based WNV subunit vaccine formulated with adjuvant was evaluated in domestic geese as a surrogate species for the endangered Nēnē, the state bird of Hawai’i. Prevention of viremia following viral infection of vaccinated birds was used as the clinical endpoint of protection. ELISA and plaque reduction neutralization tests demonstrate that significant levels of vaccine antigen-specific antibody were produced in groups of birds vaccinated with 5 or 10 μg of the WN-80E antigen formulated with ISA720 adjuvant. Moreover, after challenge with WNV, no viremia was detected in vaccinated birds, whereas viremia was detected up to 4 days after and virus was detected by oral swab for 6 days after infection among control groups. Safe and effective vaccination of managed or captive endangered bird populations will protect species with critically low numbers that could not survive the added mortality of introduced disease.

The efficacy of a new recombinant subunit West Nile virus (WNV) vaccine candidate was determined in a hamster model of meningoencephalitis. Groups of hamsters were immunized subcutaneously with a WNV recombinant envelope protein (80E) with or without WNV non-structural protein 1 (NS1) mixed with adjuvant or adjuvant alone. At 2 weeks, 6 months, and 12 months after two immunizations at 4 week intervals with the respective immunogens, groups of animals were challenged via the intraperitoneal route with a virulent strain of WNV. The two recombinant antigen preparations gave similar results; hamsters in both groups had a strong antibody response following immunization, and none of the animals became ill or developed detectable viremia after challenge with WNV at 2 weeks or 6 months post booster vaccination. In contrast, , mortality among the control animals at 2 weeks post booster challenge was 73%, and at 6 months post booster, the mortality was 53% among the control animals. When challenged 12 months after the booster vaccination, a low level viremia was detected in some of the vaccinated hamsters, and one hamster became sick, but recovered. In contrast, all of the control animals that received adjuvant only developed a viremia, and the mortality rate was 77%. These results with the recombinant subunit WNV vaccine are very encouraging and warrant further animal studies to evaluate its potential use to protect humans against WNV disease.

While several West Nile vaccines are being developed, none are yet available for humans. In this study aimed at developing a vaccine for humans, West Nile virus (WNV) envelope protein (E) and non-structural protein 1 (NS1) were produced in the Drosophila S2 cell expression system. The C-terminal 20% of the E protein, which contains the membrane anchor portion, was deleted, thus allowing for efficient secretion of the truncated protein (80E) into the cell culture medium. The proteins were purified by immunoaffinity chromatography (IAC) using monoclonal antibodies that were flavivirus envelope protein group specific (for the 80E) or flavivirus NS1 group specific (for NS1). The purified proteins were produced in high yield and used in conjunction with adjuvant formulations to vaccinate mice. The mice were tested for both humoral and cellular immune responses by a plaque reduction neutralization test and ELISA, and by lymphocyte proliferation and cytokine production assays, respectively. The results revealed that the 80E and the NS1 proteins induced both high-titered ELISA and neutralizing antibodies in mice. Splenocytes from immunized mice, cultured in vitro with the vaccine antigens as stimulants, showed excellent proliferation and production of cytokines (IFN-gamma, IL-4, IL-5, and IL-10). The level of antigen-stimulated lymphocyte proliferation and cytokine production was comparable to the level obtained from mitogen (phytohemagglutinin or pokeweed) stimulation, indicating a robust cellular response as well. These findings are encouraging and warrant further in vivo studies to determine the protective efficacy of the WNV vaccine candidate.

In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration.

Objective To determine the safety and efficacy of 3 clinically relevant vaccines in patients with systemic lupus erythematosus (SLE). Methods We studied 73 consecutive SLE patients immunized with pneumococcal, tetanus toxoid (TT), and Haemophilus influenzae type B (HIB) vaccines. Patients were evaluated preimmunization and 12 weeks postimmunization for disease activity and immunization side effects. Results Eighty-four percent of the SLE patients developed a 4-fold titer increase in response to at least 1 vaccine, with 51% developing a 2-fold titer increase with all 3 vaccines. The majority of SLE patients developed protective levels of antibody to TT (90%) and HIB (88%). Although protective antibody levels could not be determined for pneumococcus, almost half of the patients (47%) developed a 4-fold antibody response. There was a trend toward a lower antibody response in patients with active disease treated with immunosuppressive therapy. Overall lupus disease activity was unaffected by immunization. Conclusion Immunization is safe in SLE patients, with the overwhelming majority developing protective antibody levels. Therefore, SLE patients should receive immunizations according to the recommendations of the Centers for Disease Control and Prevention and the Immunization Practices Advisory Committee.

We present the case of a 28-year-old Caucasian female with common variable immunodeficiency (CVID) since age 5 who had a long history of hospitalizations for unexplained fevers and pulmonary infiltrates. The patient developed mild lymphocytosis 7 months prior to our evaluation. Flow cytometry of peripheral blood revealed an expansion of γδ T lymphocytes, mild CD4 T lymphocytopenia, and a reduced CD4/CD8 ratio (0.2). Two subpopulations of γδ T lymphocytes were found (CD3+/CD4−/CD8+, 47%; CD3+/CD4−/CD8−, 53%), the vast majority of which expressed V-δ1. An infectious cause for the patient's γδ T lymphocytosis could not be found. The sputum was chronically colonized with Staphylococcus aureus, and the organism produced TSST-1 in vitro. A bronchoalveolar lavage (BAL) revealed marked lymphocytosis, but γδ T lymphocytes were not overrepresented in the BAL. Lymphocyte functional studies revealed poor proliferative responses to mitogens and staphylococcal superantigens and diminished cytokine production. V-δ1 T lymphocytes from the patient's blood were not expanded in vitro in response to staphylococcal superantigens. TCR gene rearrangement studies confirmed the presence of Jγ and Jβ1 clonal rearrangements accounting for only a small subpopulation of the γδ T lymphocytes. These studies were repeated 5 months later and were unchanged. A bone marrow biopsy was negative for leukemia. Hence, the cause of the patient's γδ T lymphocytosis could not be determined despite evaluation for underlying malignancy, occult infection, or superantigen-driven stimulation. The patient ultimately died of progressive respiratory insufficiency. The state of current knowledge regarding γδ T lymphocytosis, decreased production of αβ T lymphocytes, and a low CD4/CD8 ratio in association with CVID is discussed.

Although West Nile Virus (WNV) has not been reported in Hawai'i, eventual introduction appears unavoidable with potential adverse effects on avian species. Nēnē (Branta sandvicensis) are endemic endangered Hawaiian geese that are susceptible to WNV. We demonstrate that a vaccine developed against WNV for humans (WN-80E) is also highly immunogenic in Nēnē and does not produce adverse biologic effects. Six captive, nonbreeding Nēnē were immunized with two 10-μg doses (4 wk apart) of the WN-80E recombinant protein adjuvanted with Montanide ISA720. Two Nēnē were similarly injected with "mock" preparation as controls. Blood samples were collected before the first dose, then 2 wk and 6 mo after the second dose. WNV-specific antibody titers were determined by an endpoint enzyme-linked immunosorbent assay. An unpaired t-test demonstrated significantly higher geometric mean titers for immunized vs. control groups 2 wk after dose 2 (4,129 and 100, respectively, P=0.010) and 6 mo after dose 2 (246 and 63, respectively, P=0.002). Daily observations revealed no swelling at the site of injection and no serious adverse biological effects from the immunization. The vaccine containing the WN-80E and Montanide ISA720 adjuvant appears to be safe and immunogenic in Nēnē. This protein-based WNV vaccine may be safer for use in Hawai'i than killed virus and live chimeric or recombinant canarypox-vectored vaccines because it cannot cause disease.

Extracts of liver and spleen were used to isolate opossum adenosine deaminase isoenzymes (ADA1 and ADA2) and to determine their activities with adenosine and 2'-deoxyadenosine as substrates. Km values (microM) for adenosine and 2'-deoxyadenosine, respectively, as substrates for partially purified opossum liver adenosine deaminase isoenzymes were ADA1: 57 +/- 7 vs. 26 +/- 4 and ADA2: 285 +/- 25 vs. 580 +/- 92. In crude spleen extract, ADA2 activity was stable at 56 degrees C during 40 min of incubation. ADA1 activity declined in a linear fashion under the above conditions with an apparent T1/2 of 80 min. Sephadex G-150 column chromatography of crude spleen extract showed the apparent molecular weight of the ADA activity not inhibited by (+/-)-EHNA (i.e. ADA2) to be 170 kDa; ADA activity fully inhibited by (+/-)-EHNA (i.e. ADA1) eluted in the fractions corresponding to a molecular weight of 35 kDa.

The efficacy of a recombinant subunit West Nile (WN) vaccine candidate was determined in a hamster model of encephalitis. Animals included young, aged, and immunocompromised animals in an effort to simulate key groups at risk of WN virus–induced disease. Groups of aged (12 month old), weanling, and adult hamsters rendered leukopenic after immunization were immunized subcutaneously with a WN virus recombinant envelope protein (WN-80E) with or without WN virus non-structural protein 1 (NS1) mixed with adjuvant or adjuvant alone. A challenge dose of wild-type WN virus was administered to produce 40–100% mortality in the control hamsters. The recombinant antigen preparations containing WN-80E with or without WN NS1 gave similar results. Hamsters in both groups had a strong antibody response after immunization, and none of the aged or weanling animals became ill or developed detectable viremia after challenge with WN virus at 2 weeks after booster vaccination. However, mortality among the control animals (administered adjuvant without antigen) at 2 weeks after booster challenge was 40–60%. In hamsters rendered leukopenic after immunization, survival rates up to 80% were observed, and a low-level viremia was detected in the vaccinated and challenged hamsters. The survival rate was significantly (P < 0.05) higher in animals vaccinated with a higher dose of WN-80E than a lower dose. The addition of NS1 did not significantly affect survival after challenge. In contrast, all of the control animals that received adjuvant only developed a very high level of viremia, and the mortality rate was 100%. These findings indicate that the recombinant WN vaccines induced antibody in and afforded protection to young and aged hamsters and immunosuppressed hamsters.

A combination of parallel chemical synthesis and biocatalysis has been used to prepare and amplify a library of cross-conjugated cyclopentenones. A number of marine and terrestrial natural products with antibiotic activity are known to incorporate this pharmacophore. The library was screened for anticancer, antimycobacterial, antifungal, and antibacterial activity. The positive results from the screens provide an indication of the structural features that are associated with activity in the various assays and suggest promising avenues for further inquiry.

The production of interleukin 2 (IL-2) gamma interferon, IL-4, tumor necrosis factor alpha (TNF-α), TNF-β, IL-5, and IL-10 in vitro by peripheral blood mononuclear cells cultured from healthy immunocompetent subjects after mitogen stimulation was determined. The mitogens used were concanavalin A, phytohemagglutinin, pokeweed mitogen, and Staphylococcus aureus Cowen. The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro.

Neutrophil activation by phorbol 12-myristate 13-acetate (PMA) and zymosan was assessed by luminol-dependent chemiluminescence (CL) in a novel microtiter plate format and by flow cytometry (FC) based on the oxidation of dihydrorhodamine 123. The results of this comparison demonstrated striking differences in kinetic parameters between these two techniques for neutrophil activation by PMA and zymosan. PMA activation, as determined by FC, was found to be an all-or-none phenomenon in that below a critical concentration of PMA, few cells were positive. Above this concentration, almost all cells were positive; however, the fluorescence intensity of positive cells increased with an increasing PMA concentration until a plateau (maximal) level was reached. In contrast, increasing zymosan concentrations resulted in proportionate increases in the percentage of positive cells until close to 100% of cells were positive. However, the fluorescence intensity of positive cells remained about the same. CL activity increased proportionately with either PMA or zymosan concentration until a maximal level was achieved. The concentration of PMA required for half-maximal activity was about 10-fold higher for FC than for CL, whereas the analogous concentration of zymosan was about 30-fold higher for CL than for FC. In addition, opsonization had only a small negative effect on the ability of zymosan to activate neutrophils, as determined by FC, whereas it had a very large enhancing effect when determined by CL. The differences in kinetic parameters of activation suggest differential sensitivity to particulate (zymosan) versus soluble (PMA) stimulants for FC and CL.

Gamma interferon (IFN-gamma) has multiple immunomodulating effects and has been postulated as a possible immunopotentiating agent for the prevention or treatment of neonatal infections. This report describes the effect of rat recombinant IFN-gamma on the oxidative burst activity and CD11b expression of neonatal and adult rat polymorphonuclear leukocytes (PMNL). Oxidative burst activity was assessed by chemiluminescence and dihydrorhodamine flow cytometry. Neonatal PMNL exhibited significantly less oxidative burst activity than did adult PMNL. IFN-gamma mildly enhanced the chemiluminescence response of PMNL from both the rat pups and adults, but this effect was not statistically significant when analyzed by a multivariate model of repeated-measures analysis of variance for both chemiluminescence and dihydrorhodamine flow cytometry. CD11b expression was also not significantly enhanced by IFN-gamma.

Pseudomonas aeruginosa ribosomal vaccines were tested for their ability to protect rats subjected to a 20% total body surface burn against the lethal effects of infection with homologous organisms. When administered prior to burning, the vaccines provided 100% protection. When administered postburning, the vaccine from one strain also provided 100% protection when the time interval between vaccination and infection was 3 days. When this time interval was reduced to 1 or 2 days, approximately 50% protection was obtained with the same vaccine. The vaccine from a second strain tested provided about 50% protection with a 3-day time interval. In addition, passive immunization using antiserum to a ribosomal vaccine was also demonstrated to be effective in protecting burned and infected rats, especially when multiple doses of antiserum were used. In this case, 80% protection was obtained (with no protection observed using multiple doses of normal serum). Finally, a comparison of ribosomal and lipopolysaccharide vaccines and antisera was also performed.

In a study of the efficacy of following up an initially negative enzyme immunosorbent assay (EIA) rapid streptococcal antigen detection test with a throat culture, 2 double swabs (4 total) were obtained from 264 pediatric patients with sore throats. Although a throat culture was more specific (97%) than the EIA (89%), the sensitivity (87%) and negative predictive value (97%) of a single EIA was the same as that of a single throat culture. A follow-up throat culture was more accurate than a follow-up EIA. We conclude that the office EIA tested results in more false positives but misses no more true positives than a single throat culture processed by a well-controlled microbiology laboratory. If a follow-up technique is used for an initially negative EIA rapid streptococcal antigen detection test, the throat culture is the superior test and would be equally applicable following an initially negative throat culture or EIA.

Two mannose-negative mutants of Escherichia coli K-12 have been isolated. These mutants are deficient in the ability to synthesize phosphomannose isomerase and capsular polysaccharide when grown on glucose-containing media. Interrupted mating experiments to determine the kinetics of genetic transfer show that the two mannose-negative mutations map together between the histidine and tryptophan regions of the E. coli chromosome.

Mutants of Escherichia coli K12 at the cap R locus are overproduce capsular polysaccharide, and are derepressed for synthesis of several enzymes involved in capsular polysaccharide synthesis 1-3 including GDP-mannose pyrophosphorylase. 8 UDP-glucose pyrophosphorylase is also derepressed in a haploid cap R9 mucoid mutant. Heterozygous mucoid partial diploids with the cap R9 allele on the episome and the wild-type ( cap R + ) allele on the chromosome (F′ cap R9/ cap R + ) are derepressed for UDP-glucose pyrophosphorylase and GDP-mannose pyrophosphorylase, while the reciprocal nonmucoid heterozygotes (F′ cap R + / cap R9) are repressed for these enzymes. These results provide evidence that the episomal cap R allele is dominant with respect to synthesis of these two enzymes.

1. Cytochrome oxidase from an extremely halophilic bacterium requires up to 5.0 M NaCl for maximal activity and stability. Differences in effectiveness among various salts in promoting enzyme activity were observed, but a minimal activity (about 30%) could be supported by all salts tested including MgCl 2 and spermine at relatively low concentrations. The enzyme activity in the presence of low concentrations of MgCl 2 was shown to be more dependent on pH but less sensitive to hydrophobic bond-breaking agents, such as ethanol and especially n -propanol, than the enzyme activity in the presence of high concentrations of NaCl. 2. Spontaneous inactivation of the enzyme in the absence of salt demonstrated that the rate of inactivation of the MgCl 2 -dependent activity has a greater temperature dependence than the rate of inactivation of the NaCl-dependent activity. The effect of temperature on the rate of inactivation of the NaCl-dependent activity was found to be greatest at neutral pH and became minimal at acid or alkaline pH. Inactivation of the enzyme in the presence of high concentrations of NaCl (by prolonged incubation) demonstrated that the enzyme was inactivated more rapidly at −10° than at 5° at pH 4.0, but not at pH 7.5. 3. These results suggest that hydrophobic forces predominate in promoting the major portion of the enzyme activity in the presence of high NaCl concentrations, while charge-shielding promotes partial enzyme activity in the presence of MgCl 2 . Hydrophobic bonds also appear to be involved in the stability of the enzyme, particularly at acid and alkaline pH, where hydrogen bonding is expected to be less extensive.

The opsonic capacity of antisera to Pseudomonas aeruginosa ribosomal vaccine fractions was determined by a chemiluminescent technique. Antiserum to a vaccine fraction ("peak A") containing lipopolysaccharide (antiserum A), and antiserum to a vaccine fraction ("peak B"), which did not contain detectable amounts of lipopolysaccharide (antiserum B), were used to opsonify live or formalin-treated bacteria. Polymorphonuclear leukocytes were then stimulated by the opsonified bacteria in the presence of the chemiluminigenic probe, luminol, resulting in the observed chemiluminescence. The data obtained indicated that the antisera had comparable opsonic activity with live (untreated) bacteria. However, antiserum B had far less opsonic activity than did antiserum A when formalinized bacteria were used. Owing to the effects of formaldehyde on protein, these results were interpreted as evidence to suggest that the opsonic activities of the two antisera are dependent on different antigens on the bacterial cell surface. Antiserum A activity is probably dependent on lipopolysaccharide to a great extent, whereas antiserum B activity is most likely dependent primarily on a protein(s).

The last few years have seen a dramatic increase in the number of rapid antigen detection tests for patients with group A fl-hemolytic streptococcal pharyngitis. In general, most of the ADTs are specific enough, in comparison with the throat culture, that a positive ADT result can stand alone. A negative ADT result, however, in the opinion of most investigators, lacks adequate sensitivity to be used without a ,backup" throat culture? 4 Because most clinical studies involve only one or two ADTs at a tim e, and because of the variety of clinical settings in which they are tested, few data exist with regard to the relative sensitivity of various ADTs tested in common settings and conditions. Moreover, it is virtually impossible to obtain meaningful comparative data from in viva sources, because no two throat swabs will yield the same number of organisms, even when obtained from the same source (Lewey S, White CB, Lieberman MM, Morales E: unpublished data). Thus we used a simple in vitro method to determine the relative sensitivity of eight commercially available rapid ADTs for the detection of serially diluted broth concentrations of GABHS.

Studies of the protective activity of a ribosomal vaccine from Pseudomonas aeruginosa in various immunocompromised animal models were performed. The results obtained demonstrated that the vaccine was highly effective in complement (C5)-deficient mice, C3-deficient (cobra venom factortreated) mice, and leukopenic mice in providing protection against lethal infection with P. aeruginosa . Passive immunization with specific antiserum to the ribosomal vaccine was also effective in leukopenic mice. In mice that were both C5-deficient and leukopenic, the vaccine did not protect mice against lethal infection, but did prolong their survival, whereas in C3-deficient, leukopenic mice significantly enhanced protection was observed. These results were interpreted to suggest the involvement of multiple factors in the protective immune response to the vaccine, including the bactericidal and opsonic activities of specific antibody plus complement and the phagocytic activity of polymorphonuclear leukocytes with opsonized bacteria. Compensation for deficiencies in some of these factors can be obtained by enhancement of other factors through active or passive immunization.

In vitro bactericidal and opsonophagocytic assays were performed with mouse antiserum to a ribosomal vaccine from Pseudomonas aeruginosa. The results obtained demonstrated that both specific antibody and complement are required for bactericidal and opsonic activities. C4-deficient complement also supported these activities. Polymorphonuclear leukocytes (PMNL) are not required, but when present, augment the observed killing activity. Opsonic and bactericidal activities were found to increase with increasing complement concentrations in an apparently logarithmic fashion until maximal activity was obtained. On the contrary, determination of bactericidal activity as a function of specific antiserum concentration revealed a "prozone" effect; i.e., maximal activity was obtained at an intermediate concentration of antiserum with significantly decreased activity at both higher and lower concentrations. This effect was not observed when immunoglobulin G (IgG) purified from specific antiserum by a method involving exposure to pH 9.0 and pH 3.0 was used instead of whole antiserum. In this case bactericidal activity increased as IgG concentration was increased, also in an apparently logarithmic manner.

The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

Mutations in a regulator gene ( capR ) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepress synthesis of guanosine diphosphate (GDP)-mannose pyrophosphorylase. In addition, a second mucoid mutation ( capS , which maps separately from capR ) also results in the derepression of GDP-mannose pyrophosphorylase. New conditions for assaying GDP-mannose hydrolyase and GDP- l -fucose synthetase permitted us to show that these enzymes are also derepressed in the capS mucoid strain. Although phosphomannose isomerase and uridine diphosphate-galactose-4-epimerase are derepressed in capR mucoid strains, they are not derepressed in capS mucoid strains. A nonmucoid mutant of a strain containing the capR9 (mucoid) allele was deficient in GDP-mannose pyrophosphorylase.

A regulator gene mutation ( capR ) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepresses phosphomannose isomerase (PMI) synthesis. In contrast, a second mutation ( capS , which maps separately from capR ) that causes increased production of the same polysaccharide does not lead to increased synthesis of PMI (nor of several of the other enzymes involved in polysaccharide synthesis). Introduction of the capR9 allele by transduction or mutation of capR + to capR can change the phenotype of a mannose-negative nonmucoid strain to a mannose-positive mucoid phenotype. Thus, genotype capR + man-2 is mannose-negative and nonmucoid, but genotype capR9 man-2 is mannose positive and mucoid. Other interactions between these alleles in the synthesis of capsular polysaccharide are recorded.

Uridine diphosphate (UDP)-glucose dehydrogenase, the enzyme that converts UDP-glucose to UDP-glucuronic acid, was derepressed in a mucoid ( capR9 ) strain of Escherichia coli K-12 and repressed in a nonmucoid ( capR + ) strain. A nonmucoid mutant (strain MC 152; capR9 non-2 ) derived from the mucoid strain accumulated large quantities of nucleotides. Among these nucleotides, UDP-glucuronic acid was identified as well as guanosine triphosphate and an adenosine diphosphate-sugar. UDP-glucose dehydrogenase was still derepressed in strain MC 152. When the nonmucoid mutant was transduced to the wild-type state for this regulator gene ( capR + ), the transductant was found to accumulate less total nucleotides, and the accumulation of UDP-glucuronic acid was abolished. UDP-glucose dehydrogenase was repressed in the capR + non-2 strain but not to the same extent that it was in the capR + strain.