Onset and duration of protective immunity to IA/IB and IE strains of Venezuelan equine encephalitis virus in vaccinated mice.

Three vaccines developed for protection against IA/IB subtypes of Venezuelan equine encephalitis (VEE) virus were evaluated in mice for the ability to protect against systemic and mucosal challenges with a virulent virus of the IE subtype. The vaccines were the formaldehyde-inactivated C-84 and live attenuated TC-83 vaccines currently administered to people under investigational new drug (IND) status, and a new live attenuated vaccine candidate, V3526. V3526 was superior for inducing protection to VEE IA/IB within a week of vaccination, and protection persisted for at least a year. All three vaccines induced long-term clinical protection against peripheral or mucosal challenge with IE virus, with the mucosal immunity induced by attenuated vaccines lasting longer than that induced by the inactivated vaccine. These data show that the molecularly cloned V3526 vaccine induces equivalent or improved immunity to homologous and heterologous VEE viruses than the existing vaccines.

Vaccine potential of Ebola virus VP24, VP30, VP35, and VP40 proteins.

Previous vaccine efforts with Ebola virus Zaire (EBOV-Z) emphasized the potential protective efficacies of immune responses to the surface glycoprotein and the nucleoprotein. To determine whether the VP24, VP30, VP35, and VP40 proteins are also capable of eliciting protective immune responses, these genes were expressed from alphavirus replicons and used to vaccinate BALB/c and C57BL/6 mice. Although all of the VP proteins were capable of inducing protective immune responses, no single VP protein protected both strains of mice tested. VP24, VP30, and VP40 induced protective immune responses in BALB/c mice, whereas C57BL/6 mice survived challenge only after vaccination with VP35. Passive transfer of immune sera to the VP proteins did not protect unvaccinated mice from lethal disease. The demonstration that the VP proteins are capable of eliciting protective immune responses to EBOV-Z indicates that they may be important components of a vaccine designed to protect humans from Ebola hemorrhagic fever.

Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein.

Cytotoxic T lymphocytes (CTLs) are proposed to be critical for protection from intracellular pathogens such as Ebola virus. However, there have been no demonstrations that protection against Ebola virus is mediated by Ebola virus-specific CTLs. Here, we report that C57BL/6 mice vaccinated with Venezuelan equine encephalitis virus replicons encoding the Ebola virus nucleoprotein (NP) survived lethal challenge with Ebola virus. Vaccination induced both antibodies to the NP and a major histocompatibility complex class I-restricted CTL response to an 11-amino-acid sequence in the amino-terminal portion of the Ebola virus NP. Passive transfer of polyclonal NP-specific antiserum did not protect recipient mice. In contrast, adoptive transfer of CTLs specific for the Ebola virus NP protected unvaccinated mice from lethal Ebola virus challenge. The protective CTLs were CD8(+), restricted to the D(b) class I molecule, and recognized an epitope within amino acids 43 to 53 (VYQVNNLEEIC) in the Ebola virus NP. The demonstration that CTLs can prevent lethal Ebola virus infection affects vaccine development in that protective cellular immune responses may be required for optimal protection from Ebola virus.

Ebola virus: the search for vaccines and treatments.

Ebola viruses belong to the family Filoviridae, which are among the most virulent infectious agents known. These viruses cause acute, and frequently fatal, hemorrhagic fever in humans and nonhuman primates. Currently, no vaccines or treatments are available for human use. This review describes Ebola viruses, with a particular focus on the status of research efforts to develop vaccines and therapeutics and to identify the immune mechanisms of protection.

Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated V3526 vaccine candidate.

The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.

Epitopes involved in antibody-mediated protection from Ebola virus.

To determine the ability of antibodies to provide protection from Ebola viruses, monoclonal antibodies (mAbs) to the Ebola glycoprotein were generated and evaluated for efficacy. We identified several protective mAbs directed toward five unique epitopes on Ebola glycoprotein. One of the epitopes is conserved among all Ebola viruses that are known to be pathogenic for humans. Some protective mAbs were also effective therapeutically when administered to mice 2 days after exposure to lethal Ebola virus. The identification of protective mAbs has important implications for developing vaccines and therapies for Ebola virus.

Cellular distributions and functions of histamine, octopamine, and serotonin in the peripheral visual system, brain, and circumesophageal ring of the horseshoe crab Limulus polyphemus.

The data reviewed here show that histamine, octopamine, and serotonin are abundant in the visual system of the horseshoe crab Limulus polyphemus. Anatomical and biochemical evidence, including new biochemical data presented here, indicates that histamine is a neurotransmitter in primary retinal afferents, and that it may be involved in visual information processing within the lateral eye. The presence of histamine in neurons of the central nervous system outside of the visual centers suggests that this amine also has functions unrelated to vision. However, the physiological actions of histamine in the Limulus nervous system are not yet known. Octopamine is present in and released from the axons of neurons that transmit circadian information from the brain to the eyes, and octopamine mimics the actions of circadian input on many retinal functions. In addition, octopamine probably has major functions in other parts of the nervous system as octopamine immunoreactive processes are widely distributed in the central nervous system and in peripheral motor nerves. Indeed, octopamine modulates functions of the heart and exoskeletal muscles as well as the eyes. A surprising finding is that although octopamine is a circulating neurohormone in Limulus, there is no structural evidence for its release into the hemolymph from central sites. The distribution of serotonin in Limulus brain suggests this amine modulates the central processing of visual information. Serotonin modulates cholinergic synapses in the central nervous system, but nothing further is known about its physiological actions.

Comparative neurovirulence and tissue tropism of wild-type and attenuated strains of Venezuelan equine encephalitis virus administered by aerosol in C3H/HeN and BALB/c mice.

To assess the potential for aerosol administration of vaccines for Venezuelan equine encephalitis virus (VEE), we compared the neurovirulence and tissue tropism of the wild-type Trinidad donkey (TrD) strain to those of the attenuated TC83 and V3526 strains of VEE in mice. Six to 8-week-old female C3H/HeN and BALB/c mice were aerosol exposed to one of the three VEE strains. Three mice of each strain were euthanatized at different times and their tissues were processed and stained using hematoxylin and eosin, immunohistochemistry, and in situ hybridization. All three viral strains infected the brains of mice and induced encephalitis. TrD spread caudally from the olfactory bulbs to all regions of the brain, caused widespread necrotizing panencephalitis by day 5, and resulted in 100% mortality (geometric mean = 7 days) in both mouse strains. By comparison, TC83 relatively spared the caudal regions of the brain but still caused 100% mortality in the C3H/HeN mice (geometric mean = 12 days), yet it did not kill any BALB/c mice. V3526 infectivity of the brain was the most limited, mainly affecting the neocortex and diencephalon. This virus was not lethal in either mouse strain. The TrD strain also infected the olfactory neuroepithelium, local lymphoid tissues, teeth, and vomeronasal organs, whereas the affinity of TC83 and V3526 outside the brain was essentially limited to the olfactory neuroepithelium. Attenuated VEE strains administered to mice by aerosol have restricted tissue tropism as compared with wild-type virus; however, even attenuated strains can infect the brain and induce encephalitis.

Venezuelan equine encephalitis virus vaccines induce mucosal IgA responses and protection from airborne infection in BALB/c, but not C3H/HeN mice.

Immunization with either a live-attenuated (TC-83) or formalin-inactivated (C-84) vaccine for Venezuelan equine encephalitis (VEE) virus protected BALB/c mice from lethal VEE infection acquired subcutaneously or by aerosol. While vaccinated C3H/HeN mice were also protected from parenteral infection, neither vaccine protected these mice from an aerosol infection. The apparent vaccine failures in C3H/HeN mice could not be attributed to deficiencies in virus-neutralizing antibodies in serum, as these responses were typically of equal or higher titer than those observed in protected BALB/c mice before challenge. IgG subclass analysis offered no facile explanation: profiles of IgG2 alpha dominance were observed in C3H/HeN mice given either vaccine and in BALB/c mice given the live-attenuated vaccine, whereas BALB/c antibody responses shifted toward IgGl dominance after immunization with the killed C-84 vaccine. Data from immunized congenic mice showed that the H-2 genes from the C3H/He mice were not singularly responsible for the inability of these mice to resist aerosol infection with VEE virus. VEE virus-specific IgA responses were detected more frequently in respiratory and vaginal secretions obtained from the protected BALB/c mice.

Mucosal immunity induced by parenteral immunization with a live attenuated Venezuelan equine encephalitis virus vaccine candidate.

Induction of a mucosal immune response is generally thought to require introduction of an immunogen directly onto the mucosal surface. It has been observed, however, that live, attenuated mutants of the alphavirus, Venezuelan equine encephalitis virus (VEE), induce protection from virulent challenge at the respiratory mucosa even after parenteral inoculation. In this report, we propose a mechanism by which subcutaneous immunization with a molecularly cloned, attenuated double mutant of VEE is able to stimulate the production of mucosal anti-VEE IgA. Our results showed that the immunizing virus spread to, and replicated within, lymphoid tissues throughout the mouse. Several tissues known to be inductive sites of the mucosal immune system were found to be positive for the presence of VEE RNA by 48 hr postimmunization. Moreover, this mucosal lymphotropism resulted in the production of virus-specific IgA antibody detectable in vaginal secretions of immunized mice.

Application of control chart statistics to blood pressure measurement variability in the primary care setting.

This study was undertaken as an interdisciplinary effort in response to a frequently frustrating clinical problem of interpreting variable blood pressure measurements under uncertain conditions of quality control. The method is an application of the Shewhart Control Chart analysis to blood pressure measurement in adults in an academic nursing center. The natural variability found in a series of blood pressure readings is measured and described after a pilot study to eliminate examiner, equipment, and time interval variability. Results revealed that there was a significant drop from the first systolic reading to subsequent readings. A three standard deviation limit will be met if the range between the second and third systolic reading does not exceed 13 and the range between the second and third diastolic readings does not exceed 11. Thus for recognition and management of measurement variability, three blood pressure measurements 1 minute apart should be taken as a routine, and the average of the second and third reading recorded. The positive impact on clients, providers, and interdisciplinary research colleagues in changing measurement technique to achieve greater quality in clinical practice is discussed. Limitations and recommendations are presented.

Conversion of an immunogenic human immunodeficiency virus (HIV) envelope synthetic peptide to a tolerogen in chimpanzees by the fusogenic domain of HIV gp41 envelope protein.

The fusogenic (F) domain of human immunodeficiency virus (HIV) gp41 envelope (env) protein has sequence similarities to many virus and mediates the fusion of HIV-infected cells. During a survey of the immunogenicity of HIV env peptides in chimpanzees, we have observed that HIV peptide immunogenicity was dramatically altered by the NH2-terminal synthesis of the gp41 F domain to an otherwise immunogenic peptide. We compared two hybrid peptide types comprised of T helper (Th) and B cell epitopes of HIV gp120 env protein for their immunogenicity in chimpanzees. The Th-B epitope hybrid peptides contained the HIV gp120 Th cell determinant, T1 (amino acids [aa] 428-440)-synthesized NH2 terminal to gp120 V3 loop peptides, which contain B cell epitopes that induce anti-HIV-neutralizing antibodies (SP10IIIB [aa 303-321] and SP10IIIB [A] [aa 303-327]). The F-Th-B peptide contained the HIV gp41 F domain of HIVIIIB gp41 (aa 519-530)-synthesized NH2 terminal to the Th-B peptide. Whereas Th-B peptides were potent immunogens for chimpanzee antibody and T cell-proliferative responses, the F-Th-B peptide induced lower anti-HIV gp120 T and B cell responses. Moreover, immunization of chimpanzees with F-Th-B peptide but not Th-B peptides induced a significant decrease in peripheral blood T lymphocytes (mean decrease during immunization, 52%; p < 0.02). Chimpanzees previously immunized with F-Th-B peptide did not respond well to immunization with Th-B peptide with T or B cell responses to HIV peptides, demonstrating that the F-Th-B peptide induced immune hyporesponsiveness to Th and B HIV gp120 env determinants. These observations raise the hypothesis that the HIV gp41 env F domain may be a biologically active immunoregulatory peptide in vivo, and by an as yet uncharacterized mechanism, promotes primate immune system hyporesponsiveness to otherwise immunogenic peptides.

Priming of anti-human immunodeficiency virus (HIV) CD8+ cytotoxic T cells in vivo by carrier-free HIV synthetic peptides.

The generation of antiviral cytotoxic T lymphocytes (CTLs) is a critical component of the immune response to viral infections. A safe and nontoxic vaccine for AIDS would optimally use a carrier-free synthetic peptide immunogen containing only components of HIV necessary for induction of protective immune responses. We report that hybrid synthetic peptides containing either a HIV envelope gp120 T-cell determinant (T1) or the envelope gp41 fusion domain (F) N-terminal to HIV CTL determinants are capable of priming murine CD8+, major histocompatibility complex class I-restricted anti-HIV CTLs in vivo. These data demonstrate that carrier-free, nonderivatized synthetic peptides can be used in vivo to induce anti-HIV CTL responses.

Induction of T cell CD7 gene transcription by nonmitogenic ionomycin-induced transmembrane calcium flux.

The CD7 molecule is a 40-kDa member of the Ig superfamily that has structural homology to the murine Thy-1 molecule and is acquired early in human T cell ontogeny. Previous studies have demonstrated that expression of the CD7 molecule is markedly up-regulated during T cell activation. In this study, we have studied the signals required for CD7 up-regulation on human T cells. We found that nonmitogenic amounts of ionomycin selectively and maximally up-regulated T cell CD7 on mature (peripheral blood) T cells after 24 h. Whereas CD7 expression was increased 78 +/- 25% by 0.5 microM ionomycin, expression of CD25 (IL-2R alpha), class II MHC, 4F2, transferrin receptor, CD2, CD3, CD4, CD5, and CD8 molecules was not increased. Ionomycin-induced CD7 surface expression was associated with peak increases in CD7 mRNA after 4 to 6 h. Transcriptional analysis and CD7 mRNA half-life determination revealed the increase in CD7 mRNA was the result of increased CD7 gene transcription 1 h after ionomycin stimulation and was not due to prolongation of CD7 mRNA half-life. The up-regulation of surface CD7 expression by ionomycin was dependent on extracellular calcium and did not require the activation of T cell tyrosine protein kinase. Mitogenic CD2 and CD3 mAb as well as stimulation of T cells by PHA also up-regulated CD7 expression. CD7 up-regulation by ionomycin was transient (24 to 72 h) and inhibitable by cyclosporin A, whereas CD7 up-regulation by PHA was sustained over 5 to 7 days and was significantly less inhibitable by cyclosporin A. These data demonstrate that induction of a transmembrane calcium flux generates signals that lead to CD7 gene transcription.

Synthetic peptides containing T and B cell epitopes from human immunodeficiency virus envelope gp120 induce anti-HIV proliferative responses and high titers of neutralizing antibodies in rhesus monkeys.

We have previously described a synthetic peptide (T1-SP10) derived from two noncontiguous regions of HTLVIIIB envelope gp120 (T1, amino acids 428-443; SP10, amino acids 303-321) that induced type-specific anti-HIV neutralizing antibodies and T cell proliferative responses against native HIV gp120 when used as a carrier-free immunogen in goats. In this study, HTLVIIIB T1-SP10 synthetic peptides were used to immunize rhesus monkeys to determine if the peptides were capable of eliciting HIV-specific neutralizing antibody and proliferative responses in primates. Four compounds (alum, polyA:polyU, threonyl-muramyldipeptide (MDP) and IFA) were also compared for efficacy as adjuvants in this system. Rhesus monkeys immunized with T1-SP10 peptides generated high titers of antibodies against the immunogens and also against HTLVIIIB gp120. Sera from all four animals given T1-SP10 in IFA or threonyl-MDP neutralized infection by HTLVIIIB and blocked virus-dependent cell fusion events. A peak neutralization titer of 1:940 was seen in one animal given IFA (19600) and a titer of 1:900 was seen in one of the monkeys (17371) given threonyl-MDP. Proliferative responses of immune rhesus PBMC to T1-SP10 appeared after the first injection. After eight immunizations, two of eight monkeys (one injected with peptides in threonyl-MDP and one given peptides in IFA) had PBMC proliferative responses to native HTLVIIIB gp120. These data demonstrate that the carrier-free T1-SP10 synthetic peptide construct can induce high titers of neutralizing anti-HIV antibody responses and PBMC proliferative responses to HIV in primates.

Air-fluidized beds or conventional therapy for pressure sores. A randomized trial.

STUDY OBJECTIVE: To compare the effectiveness and adverse effects of air-fluidized beds and conventional therapy for patients with pressure sores. DESIGN: Randomized trial with both masked and unmasked comparisons of outcome after a median follow-up of 13 days (range, 4 to 77 days). SETTING: Urban, academic referral, and primary care medical center. PATIENTS: Of 140 potentially eligible hospitalized patients with pressure sores, 72 consented to randomization; 65 (90%) completed the study. INTERVENTIONS: Thirty-one patients on air-fluidized beds (Clinitron Therapy, Support Systems International, Inc., Charleston, South Carolina) repositioned every 4 hours from 0700h to 2300h without use of other antipressure devices. Thirty-four patients on conventional therapy used an alternating air-mattress covered by a foam pad (Lapidus Air Float System, American Pharmaceal Company, Cincinnati, Ohio) on a regular hospital bed; were repositioned every 2 hours; and had elbow or heel pads as needed. Topical therapy was standardized for both groups. MEASUREMENTS AND MAIN RESULTS: Pressure sores showed a median decrease in total surface area (-1.2 cm2) on air-fluidized beds, but showed a median increase (+ 0.5 cm2) on conventional therapy; 95% confidence interval (CI) for the difference between medians, -9.2 to -0.6 cm2 (p = 0.01). Improvement, as assessed from serial color photographs by investigators masked to treatment group, occurred in 71% and 47%, respectively; 95% CI for the difference, 1% to 47% (p = 0.05). For pressure sores 7.8 cm2 or greater, outcome differences between air-fluidized beds and conventional therapy were greater: median total surface area change was -5.3 and +4.0 cm2, respectively; 95% CI for the difference, -42.2 to -3.2 cm2 (p = 0.01). Improvement rates were 62% and 29% respectively; 95% CI for difference, 1% to 65% (p = 0.05). After adjusting for other factors associated with sore outcome, the estimated relative odds of showing improvement with air-fluidized beds were 5.6-fold (95% CI, 1.4 to 21.7) greater than with conventional therapy (p = 0.01). No significant increase in adverse effects was seen with air-fluidized beds. CONCLUSIONS: Our findings suggest that air-fluidized beds are more effective than conventional therapy, particularly for large pressure sores. Studies are needed to determine the effectiveness of air-fluidized beds in long-term care settings.

Lymphocyte function-associated antigen 1 (LFA-1) and natural killer (NK) cell activity: LFA-1 is not necessary for all killer: target cell interactions.

A panel of five monoclonal antibodies detecting human lymphocyte function-associated antigen 1 (LFA-1) was generated and shown by competitive binding studies to react with at least four distinct epitopes on this molecule. The antibodies were then tested for their ability to inhibit the lytic activity of a variety of different human natural killer (NK) populations on a panel of four NK-susceptible target cells (K562, MOLT-4, HSB-2, and Jurkat). When heterogeneous NK populations derived from fresh peripheral blood and mixed-lymphocyte culture (MLC)-generated lines were used, these anti-LFA-1 monoclonal antibodies (MAbs) inhibited lysis of all four NK targets; this finding supports the notion that LFA-1 molecules play an important role in NK-mediated lysis. When tested on a cloned line of NK cells (NK 3.3), lysis of K562 was inhibited by these MAbs, but lysis of the other three targets was not affected. This represents an instance where a MAb specific for LFA-1 inhibits the lytic activity of NK cells against some but not all targets; thus the LFA-1 molecule cannot be considered under all circumstances to be an absolute requirement in NK-mediated lysis.

Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes.

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.