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The emergence of immunotherapy for the treatment of human cancers has heralded a new era in oncology, one that is making its way into the veterinary clinic. As the immune system of many animal species commonly seen by veterinarians is similar to humans, there is great hope for the translation of human therapies into veterinary oncology. The simplest approach for veterinarians would be to adopt existing reagents that have been developed for human medicine, due to the potential of reduced cost and the time it takes to develop a new drug. However, this strategy may not always prove to be effective and safe with regard to certain drug platforms. Here, we review current therapeutic strategies that could exploit human reagents in veterinary medicine and also those therapies which may prove detrimental when human-specific biological molecules are used in veterinary oncology. In keeping with a One Health framework, we also discuss the potential use of single-domain antibodies (sdAbs) derived from camelid species (also known as Nanobodies™) for therapies targeting multiple veterinary animal patients without the need for species-specific reformulation. Such reagents would not only benefit the health of our veterinary species but could also guide human medicine by studying the effects of outbred animals that develop spontaneous tumors, a more relevant model of human diseases compared to traditional laboratory rodent models.

A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Van Fossen, Grutzius, Ruby, Mourich, Cebra, Bracha, Karplus, Cooley and Mehl.)

Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder characterized by premature death from myocardial infarction or stroke. It is caused by de novo single-nucleotide mutations in the LMNA gene that activate a cryptic splice donor site, resulting in the production of a toxic form of lamin A, which is termed progerin. Here we present a potential genetic therapeutic strategy that utilizes antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to block pathogenic splicing of mutant transcripts. Of several candidates, PPMO SRP-2001 provided the most significant decrease in progerin transcripts in patient fibroblasts. Intravenous delivery of SRP-2001 to a transgenic mouse model of HGPS produced significant reduction of progerin transcripts in the aorta, a particularly critical target tissue in HGPS. Long-term continuous treatment with SRP-2001 yielded a 61.6% increase in lifespan and rescue of vascular smooth muscle cell loss in large arteries. These results provide a rationale for proceeding to human trials.

Ebola virus causes an acute hemorrhagic fever lethal in primates and rodents. The contribution of host immune factors to pathogenesis has yet to be determined and may reveal efficacious targets for potential treatment. In this study, we show that the interleukin (IL)-10 signaling pathway modulates Ebola pathogenesis. IL-10(-/-) mice and wild-type mice receiving antisense targeting IL-10 signaling via disrupting expression through aberrant splice altering were resistant to ebola virus infection. IL-10(-/-) mice exhibited reduced viral titers, pathology, and levels of IL-2, IL-6, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-1 α and increased interferon (IFN)-γ relative to infected wild-type mice. Furthermore, antibody depletion studies in IL-10(-/-) mice suggest a requirement for natural killer cells and IFN-γ for protection. Together, these data demonstrate that resistance to ebola infection is regulated by IL-10 and can be targeted in a prophylactic manner to protect against lethal hemorrhagic virus challenge.

Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.

Phosphorodiamidate morpholino oligomers (PMO) are neutrally charged, sequence-specific antisense agents that interfere with targeted gene expression. PMO have been shown to be highly specific and potent therapies after cellular uptake, yet methods to detect PMO in tissue and inside the cell are limited. We offer in this report novel methods for the detection of cellular resident PMO using flow cytometry-fluorescence in situ hybridization (flow FISH) and a sandwich hybridization technique to quickly and sensitively quantify tissue resident PMO. These methods rely on oligonucleotide probes complementary to a PMO to specifically detect and quantify cell-associated and tissue resident PMO after in vitro or in vivo administration. Using the sandwich hybridization technique, we show that intranasally delivered PMO demonstrates zero-order clearance kinetics from the lung. Furthermore, PMO was detected in nonhematopoietic and hematopoietic cells of the lung regardless of influenza virus infection, although an increase in PMO uptake in infected hematopoietic cells was observed. Coincident measurement of target knock-down to cell-associated influenza A PMO concentration allowed for the calculation of an EC50.

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever in humans. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. Preclinical testing for development of antiviral or therapeutics is in part hampered due to a lack of an immunologically well-defined rodent model that exhibits similar acute hemorrhagic illness or sequelae compared to the human disease. We have identified the FVB mouse strain, which succumbs to a hemorrhagic fever-like illness when infected with lymphocytic choriomeningitis virus (LCMV). FVB mice infected with LCMV demonstrate high mortality associated with thrombocytopenia, hepatocellular and splenic necrosis, and cutaneous hemorrhage. Investigation of inflammatory mediators revealed increased IFN-γ, IL-6 and IL-17, along with increased chemokine production, at early times after LCMV infection, which suggests that a viral-induced host immune response is the cause of the pathology. Depletion of T cells at time of infection prevented mortality in all treated animals. Antisense-targeted reduction of IL-17 cytokine responsiveness provided significant protection from hemorrhagic pathology. F1 mice derived from FVB×C57BL/6 mating exhibit disease signs and mortality concomitant with the FVB challenged mice, extending this model to more widely available immunological tools. This report offers a novel animal model for arenavirus research and pre-clinical therapeutic testing.

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

Antisense technologies are widely used for the inhibition of gene expression. Although traditionally the AUG start codon of the open reading frame is targeted to disrupt ribosome assembly and initiation, an emerging approach is targeting sequences to disrupt pre-mRNA splicing. The primary advantage to using this approach is a positive read-out for an antisense effect through detection of a novel splice product, but additional benefit can be found in generating a novel splice product with altered functional properties. The antisense compounds used here are phosphorodiamidate morpholino oligomers conjugated to an arginine-rich cell penetrating peptide (P-PMO). We describe a five-step process for selecting the best candidate antisense compound for altering IL-12Rb2 expression including (1) detecting mRNA splice products by RT-PCR, (2) measuring protein expression, (3) evaluating protein function, (4) checking cellular viability, and (5) validating efficacy of the final candidate compound. The significance of targeting exons composed of a number of base pairs divisible by 3 is also discussed. The five steps described here for selecting the best candidate P-PMO to alter IL-12Rb2 expression should be applied for designing and screening antisense compounds for other gene targets.

Currently, no vaccines or therapeutics are licensed to counter Ebola or Marburg viruses, highly pathogenic filoviruses that are causative agents of viral hemorrhagic fever. Here we show that administration of positively charged phosphorodiamidate morpholino oligomers (PMOplus), delivered by various dosing strategies initiated 30-60 min after infection, protects>60% of rhesus monkeys against lethal Zaire Ebola virus (ZEBOV) and 100% of cynomolgus monkeys against Lake Victoria Marburg virus (MARV) infection. PMOplus may be useful for treating these and other highly pathogenic viruses in humans.

Contact dermatitis is the result of inflammatory responses mediated by hapten-specific activated CD8+ and CD4+ T cells. Activation-induced cell death (AICD) is a naturally occurring process regulating the resolution of T-cell responses through decreased expression of the antiapoptotic molecule cellular FLICE inhibitory protein (cFLIP). We show that targeting cFLIP expression in vitro and in vivo, with morpholino antisense applied systemically or topically in conjunction with antigen, sensitizes T cells to undergo "early" AICD resulting in tolerance. Analysis of antisense-treated CD8+ OT-1 splenocytes after co-culture with SIINFEKL-pulsed DCs showed apoptosis occurring in a dose-dependent manner with respect to cFLIP peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) concentration. A transplant acceptance model using male DO.11 donor cells and female BALB/c recipient mice showed that cFLIP antisense treatment could promote antigen tolerance. Hypersensitivity responses induced in mice by the epicutaneous application of the haptens FITC and oxazolone confirmed that topically applied cFLIP antisense could reduce inflammation. Treatment of the skin produced significant reduction in dermatitis and localized infiltration of lymphocytes. Moreover, the treatment was target- and antigen-specific, dose-dependent, and capable of inducing long-lived tolerance. These data suggest that the targeted expression of immune-regulating molecules is possible through the application of antisense to the skin.

The ligand-activated transcription factor, aryl hydrocarbon receptor (AHR), is a novel inducer of adaptive Tregs. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most potent AHR ligand, induces adaptive CD4+CD25+ Tregs during an acute graft-versus-host (GvH) response and prevents the generation of allospecific cytotoxic T lymphocytes. TCDD also suppresses the induction of experimental autoimmune encephalitis in association with an expanded population of Foxp3+ Tregs. In this study, we show that chronic treatment of NOD mice with TCDD potently suppresses the development of autoimmune Type 1 diabetes in parallel with greatly reduced pancreatic islet insulitis and an expanded population of CD4+CD25+Foxp3+ cells in the pancreatic lymph nodes. When treatment with TCDD was terminated after 15 weeks (23 weeks of age), mice developed diabetes over the next 8 weeks in association with lower numbers of Tregs and decreased activation of AHR. Analysis of the expression levels of several genes associated with inflammation, T-cell activation and/or Treg function in pancreatic lymph node cells failed to reveal any differences associated with TCDD treatment. Taken together, the data suggest that AHR activation by TCDD-like ligands may represent a novel avenue for treatment of immune-mediated diseases.

Alternative splicing of pre-mRNA leads to variation in the exons that form the mRNA, and provides eukaryotic organisms with an additional qualitative control of gene expression. Disruptions in the regulation of pre-mRNA splicing caused by heritable genomic mutations or quantitative shifts in the regulation of exon inclusion can lead to disease. Alternative exon inclusion (pre-mRNA splicing) produces different proteins with alternative activities, derived from the same pre-mRNA, and is utilized by the immune system to expand gene function. Recent advances in the delivery of splice switching oligomers to lymphoid cells, combined with the ability to manipulate mRNA splicing to either correct mis-splicing or to alter the balance of different splice forms, holds great promise for the development of new therapeutic strategies for the treatment of immune-related disease. Antisense-based targeted manipulation of various immune modulating molecules as therapeutic approaches are discussed in this review.

Although the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated through binding and activation of the aryl hydrocarbon receptor (AhR), the subsequent biochemical and molecular changes that confer immune suppression are not well understood. Mice exposed to TCDD during an acute B6-into-B6D2F1 graft-vs-host response do not develop disease, and recently this has been shown to correlate with the generation of CD4(+) T cells that express CD25 and demonstrate in vitro suppressive function. The purpose of this study was to further characterize these CD4(+) cells (TCDD-CD4(+) cells) by comparing and contrasting them with both natural regulatory CD4(+) T cells (T-regs) and vehicle-treated cells. Cellular anergy, suppressive functions, and cytokine production were examined. We found that TCDD-CD4(+) cells actively proliferate in response to various stimuli but suppress IL-2 production and the proliferation of effector T cells. Like natural T-regs, TCDD-CD4(+) cells do not produce IL-2 and their suppressive function is contact dependent but abrogated by costimulation through glucocorticoid-induced TNFR (GITR). TCDD-CD4(+) cells also secrete significant amounts of IL-10 in response to both polyclonal and alloantigen stimuli. Several genes were significantly up-regulated in TCDD-CD4(+) cells including TGF-beta3, Blimp-1, and granzyme B, as well as genes associated with the IL12-Rb2 signaling pathway. TCDD-CD4(+) cells demonstrated an increased responsiveness to IL-12 as indicated by the phosphorylation levels of STAT4. Only 2% of TCDD-CD4(+) cells express Foxp3, suggesting that the AhR does not rely on Foxp3 for suppressive activity. The generation of CD4(+) cells with regulatory function mediated through activation of the AhR by TCDD may represent a novel pathway for the induction of T-regs.

New methods to combat influenza A virus (FLUAV) in humans and animals are needed. The H3N8 subtype virus was the cause of the pandemic of 1890 and has recently undergone cross-species transmission from horses to dogs in the USA. In 2007 H3N8 spread to Australia, a continent previously devoid of equine influenza. Here, we show that antisense-peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), delivered by intranasal administration, are able to inhibit the replication of FLUAV A/Eq/Miami/1/63 (H3N8) in mice by over 95% compared to controls. Monitoring of body weight and immune cell infiltrates in the lungs of noninfected mice indicated that PPMO treatment was not toxic at a concentration shown to be effectively antiviral in vivo. In addition, we detected a naturally occurring mutation within the PPMO target site of a viral gene that may be the cause of resistance to one of the two antisense PPMO sequences tested. These data indicate that PPMOs targeting highly conserved regions of FLUAV are promising novel therapeutic candidates.

Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR)(4) peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time- and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.

Antisense oligomers have been shown to be effective tools for inhibiting gene expression in a highly specific manner. This technology has proven to be invaluable for determining gene function in conventional molecular and cellular studies. However, the promise of an antisense-based drug technology, suggested by antiviral efficacy shown nearly 25 years ago, is just now coming of age. Since then, numerous antisense approaches have been shown to be effective in animal models against numerous viruses and some tumors. Not surprisingly, antisense agents targeting these diseases are taking the lead in human clinical trials and FDA approval. Although comparatively smaller in scope, approaches for modulating immune responses to treat Crohn's disease, diabetes, multiple sclerosis and transplant rejection appear to be the next burgeoning phase of development in antisense therapy.

Activation of the aryl hydrocarbon receptor (AhR) by its most potent ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to immune suppression in mice. Although the underlying mechanisms responsible for AhR-mediated immune suppression are not known, previous studies have shown that activation of the AhR must occur within the first 3 days of an immune response and that CD4+ T cells are primary targets. Using the B6-into-B6D2F1 model of an acute graft-vs-host response, we show that activation of AhR in donor T cells leads to the generation of a subpopulation of CD4+ T cells that expresses high levels of CD25, along with CD62L(low), CTLA-4, and glucocorticoid-induced TNFR. These donor-derived CD4+ CD25+ cells also display functional characteristics of regulatory T cells in vitro. These findings suggest a novel role for AhR in the induction of regulatory T cells and provide a new perspective on the mechanisms that underlie the profound immune suppression induced by exposure to TCDD.

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.

4-1BB (CD137) is a tumour necrosis factor receptor (TNFR) family member, expressed primarily on CD8 T cells after activation. Signalling through 4-1BB has been reported to enhance CD8 T-cell expansion and to protect activated CD8 T cells from death, resulting in an enlarged memory population. Although stimulating 4-1BB has been shown to significantly improve the immune response to weak immunogens such as tumours, little is known about its effect on the CD8 T-cell response to a powerful viral vector such as vaccinia. To test 4-1BB's ability to improve the murine CD8 T cell response to a DNA prime, poxvirus boost vaccine, similar to those used for human immunodeficiency virus and simian immunodeficiency virus vaccines, we administered 4-1BB agonist antibody at the time of the poxvirus boost. 4-1BB stimulation increased the number of functional memory CD8 T cells by two- to fourfold. However, we saw a similar enhancement at the peak of the response and in the memory phase, thus we found no evidence in the context of virus infection that 4-1BB stimulation could increase the percentage of CD8 T cells that survive the acute activation phase to become memory cells. OX40 (CD134) is an analogous TNFR family member expressed primarily on activated CD4 T cells. OX40 stimulation increased the number of antigen-specific CD4 T cells approximately threefold. Stimulating both 4-1BB and OX40 enhanced the CD8 T-cell response more than 4-1BB alone. Thus stimulating these receptors can improve the response to a powerful virus vector, and may be useful in vaccine development.

Human chorionic gonadotropin (hCG) is expressed by many histological types of cancer and may play an important role in tumor maintenance and progression. Vaccination of patients with the therapeutic peptide Avicine (CTP37; AVI BioPharma Inc/SuperGen Inc), that contains 37 amino acids from the carboxyl terminus (CTP37; AVI BioPharma Inc/SuperGen Inc) of hCG, can result in two distinct antibody responses to separate epitopes within the peptide. Colorectal cancer patients who develop both anti-hCG responses show a significant improvement in median survival time. These observations provide a compelling rationale for the development of two human monoclonal antibodies (mAbs), one for each of the epitopes within the 37 amino acid peptide region of hCG. Two such human mAbs, both exhibiting a high degree of specificity and affinity have been prepared using XenoMouse technology. These mAbs may prove useful in multiple clinical settings for the treatment of various cancers. Treatment options may include passive immunotherapy with both mAbs, mixed passive supplement to active specific immunotherapy with Avicine and conjugation of the mAbs with radioisotopes or cytotoxic drugs. The requirement for dual mAb therapy is consistent with current trends in the development of complex, non-toxic therapies for cancer.

Plasmid DNA inoculations can induce both humoral and cellular immunity, and this technique is now being employed in developing vaccination regimens for a large number of applications. DNA vaccination studies require the preparation of large amounts of purified plasmid DNA with low endotoxin contamination, and the cost burden for multiple injections, multiple animal or large animal studies is significant. We recently reported that selective compaction with spermine can be used to purify large quantities of DNA. We wanted to determine whether this method would produce DNA suitable for vaccination. Endotoxin levels for spermine-compacted DNA were 0.3+/-0.01 endotoxin units (EU)/microg, well within the accepted range (less than 3 EU/microg) for in vivo use. When injected intramuscularly into mice, column-purified and spermine-compacted DNA induced an equivalent antigen-specific CD8+ T-cell response. The labor and time involved in purifying 5 mg of DNA by each method were similar, but the cost of spermine-compacted DNA was only 20% of the cost of column-purified DNA. We conclude that spermine compaction is an efficient and economical method for preparing vaccination-grade DNA.

Sequences of the human immunodeficiency virus (HIV) nef gene in virus isolates from 12 long-term survivors (LTSs) and 7 progressors were compared to determine if any association existed between the sequences and the corresponding clinical status. The sequences of at least five clones were determined for each subject. Conceptual translations of the open reading frames (ORFs) were examined with respect to a consensus with a prototypic nef sequence (HIV-1SF2) and for conservation of functionally described motifs. Premature stops were observed at equivalent, yet low, frequencies among the different clinical groups: 2 of 60 (3.33%) and 1 of 45 (2.22%) respectively. No remarkable differences in protein motifs implicated in several activities ascribed to Nef were noted. An association between nef sequence characteristics and the clinical state was not found.

All human immunodeficiency virus (HIV) isolates can grow readily in primary CD4(+) T cells, but they can be distinguished by their ability to replicate in macrophages and established T-cell lines. The macrophage-tropic viruses are generally non-syncytium inducing (NSI), whereas the T-cell-line-tropic viruses are syncytium inducing (SI) in cultured cells. We now demonstrate that infection of CD4(+) T cells by NSI and SI viruses shows a differential effect on production of beta-chemokines and gamma interferon. Infection by NSI viruses increased production of MIP-1alpha, MIP-1beta, and gamma interferon, whereas infection by SI viruses had no effect or decreased production of these cytokines. Production of RANTES was slightly increased during infection by both virus phenotypes. This differential effect of NSI and SI viruses was observed at the level of beta-chemokine mRNA as well as at the level of protein expression. Infection by NSI viruses also increased CD4(+) cell proliferation. These results may have relevance for a differential role of HIV strains in AIDS pathogenesis.

The presence of human herpesvirus 8 (HHV-8) was determined by polymerase chain reaction (PCR) in nasal secretions and saliva from 14 HHV-8-seropositive persons, including 8 Kaposi's sarcoma patients: 7 were human immunodeficiency virus type 1-infected, 6 of whom were asymptomatic. HHV-8 was detected in one or both body fluids in 8 (57%) of 14 subjects. Parallel PCR testing revealed the concomitant presence of cytomegalovirus, Epstein-Barr virus, and HHV-6 in various combinations in these body fluids. These data indicate frequent shedding of multiple herpesviruses in nasal secretions and saliva, particularly in Kaposi's sarcoma patients. Both body fluids are therefore potential sources HHV-8 by nonsexual transmission.

Plasmid vectors encoding the infectious hematopoietic necrosis virus (IHNV) nucleoprotein or glycoprotein gene under the control of a cytomegalovirus promoter were used to immunize rainbow trout (Oncorhynchus mykiss) against IHNV. The plasmid DNA was injected into the skeletal muscle of rainbow trout fry, and immunization was determined by the detection of virus-neutralizing and enzyme-linked immunosorbent assay antibody activity, and by protection against live virus challenge. Fish injected with the glycoprotein-encoding plasmid pCMV4-G, either alone or in combination with the nucleoprotein-encoding plasmid pCMV4-N, generated glycoprotein-specific and virus-neutralizing antibody responses. The vaccinated fish were also protected from subsequent IHNV challenge. Fish receiving pCMV4-N alone did not produce measurable virus-specific antibody and were killed by IHNV infection. These studies show that DNA vaccination will protect rainbow trout against the lethal effects of IHNV infection.

Expression of the firefly luciferase gene under the control of viral or fish promoters was observed in fish tissue after direct DNA injection of plasmid DNA. Plasmid DNA containing the firefly luciferase gene was injected into the skeletal muscle of rainbow trout (Oncorhynchus mykiss), and levels of luciferase activity were found to be dependent on the controlling promoter and the amount of injected DNA. Plasmids using the cytomegalovirus immediate early promoter (CMV-IEP) consistently produced the highest levels of luciferase activity. Maximal activity was observed five to seven days postinjection with 50 micrograms of DNA. This activity persisted in the tissues for as long as 115 days postinjection. When the DNA was examined up to two months postinjection, the predominant form was unreplicated, unintegrated DNA in linear and relaxed circular conformation. Expression of injected DNA was found predominantly within muscle cells along the injection path and in scattered muscle cells anterior to the injection site.

A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli. A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter. The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots. A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs). The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R. D. Gilmore, Jr., H. M. Engelking, D. S. Manning, and J. C. Leong. Bio/Technology 6:295-300, 1988). Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein. All fusion proteins were tested as vaccines in rainbow trout fry. Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine.