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Epstein-Barr virus (EBV) was the first human tumor virus being discovered and remains to date the only human pathogen that can transform cells in vitro. 55 years of EBV research have now brought us to the brink of an EBV vaccine. For this purpose, recombinant viral vectors and their heterologous prime-boost vaccinations, EBV-derived virus-like particles and viral envelope glycoprotein formulations are explored and are discussed in this review. Even so, cell-mediated immune control by cytotoxic lymphocytes protects healthy virus carriers from EBV-associated malignancies, antibodies might be able to prevent symptomatic primary infection, the most likely EBV-associated pathology against which EBV vaccines will be initially tested. Thus, the variety of EBV vaccines reflects the sophisticated life cycle of this human tumor virus and only vaccination in humans will finally be able to reveal the efficacy of these candidates. Nevertheless, the recently renewed efforts to develop an EBV vaccine and the long history of safe adoptive T cell transfer to treat EBV-associated malignancies suggest that this oncogenic γ-herpesvirus can be targeted by immunotherapies. Such vaccination should ideally implement the very same immune control that protects healthy EBV carriers.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with Congenital Zika Syndrome (CZS), reflecting a wide range of congenital abnormalities in fetuses and infants infected with ZIKV before birth. ZIKV infections have also been associated with the neurological autoimmune disorder known as Guillian-Barré syndrome (GBS). To date, no vaccines or antiviral strategies are licensed for ZIKV. We used rational design to develop a novel ZIKV vaccine candidate using a Woodchuck Hepatitis core Antigen (WHcAg) Virus-Like Particle (VLP) scaffold for displaying selected antigens from the ZIKV Envelope (E) protein. A Zika-VLP vaccine candidate containing the CD Loop sub-structural domain from ZIKV E protein Domain III (WHcAg CD Loop) elicited a strong immune response in a murine model. Analysis of serum immunoglobulins demonstrated induction of both Th1- and Th2- mediated immune response. No cross-reacting antibodies were detected between Zika, dengue and yellow fever virus, demonstrating a high level of specificity for the ZIKV CD Loop antigen. Immunization with the WHcAg CD Loop vaccine candidate demonstrated immunoprotection in a murine model of ZIKV infection, stimulating protective antibodies associated with antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. The WHcAg CD Loop candidate may represent a safer vaccine for preventing antibody dependent enhancement (ADE)., Competing Interests: Declaration of competing interest, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Equid alphaherpesvirus 1 (EHV-1) infection causes abortion, respiratory disease, perinatal deaths and neurological disorders in horses. The natural infection and available vaccines provide only partial and short-lived protection against reinfections. In the present study, we analyzed the ability of purified baculovirus-expressed glycoprotein D (gD) administered by different routes to induce protective immunity in BALB/c mice after challenge with the EHV-1 AR8 strain. Clinical signs varied among the different groups of mice immunized by parenteral routes, and, although gD induced a specific serum IgG response, it did not prevent the virus from reaching the lungs. Intranasally immunized mice showed no clinical signs, and virus isolation from lungs, histological lesions and antigen detection by immunohistochemistry were negative. In addition, by this route, gD did not stimulate the production of serum IgG and IgA. However, a specific IgA response in the respiratory tract was confirmed in intranasally immunized mice. Thus, we conclude that the mucosal immune response could reduce the initial viral attachment and prevent the virus from reaching the lungs. Our findings provide additional data to further study new immunization strategies in the natural host., (Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Despite recent improvements in the engineering of viral envelope proteins, it remains a significant challenge to create lentiviral vectors that allow targeted transduction to specific cell populations of interest. In this study, we developed a simple 'plug and play' strategy to retarget lentiviral vectors to any desired cell types through in vitro covalent modification of the virions with specific cell-targeting proteins (CTPs). This strategy exploits a disulfide bond-forming protein-peptide pair PDZ1 and its pentapeptide ligand (ThrGluPheCysAla, TEFCA). PDZ1 was incorporated into an engineered Sindbis virus envelope protein (Sind-PDZ1) and displayed on lentiviral particles while the TEFCA pentapeptide ligand was genetically linked to the CTP. Her2/neu-binding designed ankyrin repeat proteins (DARPin) were used as our model CTPs. DARPin-functionalized unconcentrated lentiviral vectors harboring Sind-PDZ1 envelope protein (Sind-PDZ1-pp) exhibited >800-fold higher infectious titer in HER2 + cells than the unfunctionalized virions (8.5 × 10 6 vs. <10 4 IU/mL). Moreover, by virtue of the covalent disulfide bond interaction between PDZ1 and TEFCA, the association of the CTP with the virions is nonreversible under non-reducing conditions (e.g. serum), making these functionalized virions potentially stable in an in vivo setting.

Retroviruses including Human Endogenous Retroviruses (HERVs), contain a conserved region with highly immunomodulatory functions in the transmembrane proteins in envelope gene (env) named immunosuppressive domain (ISU). In this report, we demonstrate that Env59-GP3 peptide holds therapeutic potential in a mouse model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). The results show that this specific HERV-H derived ISU peptide, but not peptide derived from another env gene HERV-K, decreased the development of EAE in C57BL/6 mice, accompanied by reduced demyelination and inhibition of inflammatory cells. Moreover, here we tested the effect of peptides on macrophages differentiation. The treatment with Env59-GPS peptide modulate the pro-inflammatory M1 profile and anti-inflammatory M2 macrophages, being shown by inhibiting inflammatory M1 hallmark genes/cytokines expression and enhancing expression of M2 associated markers. These results demonstrate that Env59-GP3 ISU peptide has therapeutic potential in EAE possibly through inducing the polarization of M2 macrophages and inhibiting inflammatory responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recently, progress has been made in the development of vaccines and monoclonal antibody cocktails that target the Ebola coat glycoprotein (GP). Based on the mutation rates for Ebola virus given its natural sequence evolution, these treatment strategies are likely to impose additional selection pressure to drive acquisition of mutations in GP that escape neutralization. Given the high degree of sequence conservation among GP of Ebola viruses, it would be challenging to determine the propensity of acquiring mutations in response to vaccine or treatment with one or a cocktail of monoclonal antibodies. In this study, we analyzed the mutability of each residue using an approach that captures the structural constraints on mutability based on the extent of its inter-residue interaction network within the three-dimensional structure of the trimeric GP. This analysis showed two distinct clusters of highly networked residues along the GP 1 -GP 2 interface, part of which overlapped with epitope surfaces of known neutralizing antibodies. This network approach also permitted us to identify additional residues in the network of the known hotspot residues of different anti-Ebola antibodies that would impact antibody-epitope interactions.

Increasing evidence shows that CC-chemokines promote inflammatory-driven angiogenesis, with little to no effect on hypoxia-mediated angiogenesis. Inhibition of the CC-chemokine class may therefore affect angiogenesis differently depending on the pathophysiological context. We compared the effect of CC-chemokine inhibition in inflammatory and physiological conditions. In vitro , the broad-spectrum CC-chemokine inhibitor "35K" inhibited inflammatory-induced endothelial cell proliferation, migration, and tubulogenesis, with more modest effects in hypoxia. In vivo , adenoviruses were used to overexpress 35K (Ad35K) and GFP (AdGFP, control virus). Plasma chemokine activity was suppressed by Ad35K in both models. In the periarterial femoral cuff model of inflammatory-driven angiogenesis, overexpression of 35K inhibited adventitial neovessel formation compared with control AdGFP-infused mice. In contrast, 35K preserved neovascularization in the hindlimb ischemia model and had no effect on physiological neovascularization in the chick chorioallantoic membrane assay. Mechanistically, 2 key angiogenic proteins (VEGF and hypoxia-inducible factor-1α) were conditionally regulated by 35K, such that expression was inhibited in inflammation but was unchanged in hypoxia. In conclusion, CC-chemokine inhibition by 35K suppresses inflammatory-driven angiogenesis while preserving physiological ischemia-mediated angiogenesis via conditional regulation of VEGF and hypoxia-inducible factor-1α. CC-chemokine inhibition may be an alternative therapeutic strategy for suppressing diseases associated with inflammatory angiogenesis without inducing the side effects caused by global inhibition.- Ridiandries, A., Tan, J. T. M., Ravindran, D., Williams, H., Medbury, H. J., Lindsay, L., Hawkins, C., Prosser, H. C. G., Bursill, C. A. CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis., (© FASEB.)

Classical swine fever (CSF) is a highly contagious swine disease caused by classical swine fever virus (CSFV). Previously, we demonstrated that rAdV-SFV-E2, an adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine against CSF, is able to protect pigs against lethal CSFV challenge. From an economical point of view, it will be beneficial to reduce the minimum effective dose of the vaccine. This study was designed to test the adjuvant effects of Salmonella enteritidis-derived bacterial ghosts (BG) to enhance the protective immunity of rAdV-SFV-E2 in pigs. Groups of 5-week-old pigs (n = 4) were immunized intramuscularly twice with 10(5) median tissue culture infective doses (TCID50) rAdV-SFV-E2 combined with 10(10) colony forming units (CFU) BG, 10(6) or 10(5) TCID50 rAdV-SFV-E2 alone or 10(10) CFU BG alone at an interval of 3 weeks, and challenged with the highly virulent CSFV Shimen strain at 1 week post-booster immunization. The results show that the pigs inoculated with 10(5) TCID50 rAdV-SFV-E2 plus BG or 10(6) TCID50 rAdV-SFV-E2 alone were completely protected from lethal CSFV challenge, in contrast with the pigs vaccinated with 10(5) TCID50 rAdV-SFV-E2 or BG alone, which displayed partial or no protection following virulent challenge. The data indicate that BG are a promising adjuvant to enhance the efficacy of rAdV-SFV-E2 and possibly other vaccines.

Although counteracting innate defenses allows oncolytic viruses (OVs) to better replicate and spread within tumors, CD8(+) T-cells restrict their capacity to trigger systemic anti-tumor immune responses. Herpes simplex virus-1 (HSV-1) evades CD8(+) T-cells by producing ICP47, which limits immune recognition of infected cells by inhibiting the transporter associated with antigen processing (TAP). Surprisingly, removing ICP47 was assumed to benefit OV immuno-therapy, but the impact of inhibiting TAP remains unknown because human HSV-1 ICP47 is not effective in rodents. Here, we engineer an HSV-1 OV to produce bovine herpesvirus UL49.5, which unlike ICP47, antagonizes rodent and human TAP. Significantly, UL49.5-expressing OVs showed superior efficacy treating bladder and breast cancer in murine models that was dependent upon CD8(+) T-cells. Besides injected subcutaneous tumors, UL49.5-OV reduced untreated, contralateral tumor size and metastases. These findings establish TAP inhibitor-armed OVs that evade CD8(+) T-cells as an immunotherapy strategy to elicit potent local and systemic anti-tumor responses.

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease of pigs. Safer and more effective vaccines are urgently needed. In this study, a synthetic ORF5 gene of porcine reproductive and respiratory syndrome virus (PRRSV) was adsorbed onto poly(D, L-lactide-co-glycolide)/polyethylenimine (PLGA/PEI) nanoparticles. We prepared a PLGA-nanoparticle-adsorbed PRRSV DNA vaccine and a PEI-DNA complex. The results showed that these model vaccines could significantly enhance humoral and cellular immune responses when compared with the responses induced by pcDNA3.1-SynORF5, a plasmid construct for expression of PRRSV ORF5. PLGA-branched PEI nanoparticles induced the most efficient immune response. The delivery system and adjuvant provide new models for the development of vaccines against PRRSV.

Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand - a single-chain antibody (scFv) to the cancer-specific HER2 receptor - expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2-gH chimera into HER2+ cells occurred in the absence of gD receptors, or upon deletion of key residues in gD that constitute the nectin1/HVEM binding sites. In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces reproductive failure in sows and respiratory problems in pigs of all ages. Live attenuated and inactivated vaccines are used on swine farms to control PRRSV. However, their protective efficacy against field strains of PRRSV remains questionable. New vaccines have been developed to improve the efficacy of these traditional vaccines. In this study, virus-like particles (VLPs) composed of the GP5 and M proteins of PRRSV were developed, and the capacity of the VLPs to elicit antigen-specific immunity was evaluated. Serum antibody titers and production of cytokines were measured in BALB/C mice immunized intramuscularly three times with different doses (0.5, 1.0, 2.0, and 4.0 μg) of the VLP vaccine. A commercial vaccine consisting of inactivated PRRSV and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. IgG titers to GP5 were significantly higher in all groups of mice vaccinated with the VLPs than in control mice. Neutralizing antibodies were only detected in mice vaccinated with 2.0 and 4.0 μg of the VLPs. Cytokine levels were determined in cell culture supernatants after in vitro stimulation of splenocytes with the VLPs for 3 days. Mice immunized with 4.0 μg of the VLPs produced a significantly higher amount of interferon-gamma (IFN-γ) than mice immunized with the commercial inactivated PRRSV vaccine and PBS. In contrast, immunization with the commercial vaccine induced higher production of IL-4 and IL-10 in mice than mice vaccinated with VLPs. These data together demonstrate the capacity of VLPs to induce both neutralizing antibodies and IFN-γ in immunized mice. The VLP vaccine developed in this study could serve as a platform for the generation of improved VLP vaccines to control PRRSV.

The purpose of this study was to investigate the potential of intranasal immunization with non-ionic surfactant vesicles (NISV) containing either the secretory recombinant form of glycoprotein B (gBs) of herpes simplex virus type 1 or a related polylysine reach peptides (DTK) for induction of protective immunity against genital herpes infection in mice. NISV were prepared by lipid film hydration method. The mean diameter of vesicles was around 390 nm for DTK-containing NISV (DTK-NISV) and 320 nm for gB1s-containing NISV (gB1s-NISV). The encapsulation efficiency of the molecules was comprised between 57% and 70%. After 7-14 day NISV maintained stable dimensions and a drug encapsulation higher than 48%. We showed that intranasal immunization with gB1s-NISV induces gB-specific IgG antibody and lymphoproliferative responses, whereas vaccination with DTK-NISV was not able to generate a gB-specific immune response. Our results indicate that vaccination of BALB/c mice with gB1s-NISV induced Th1 responses, as characterized by increased titre of IG2a in plasma and IFN-production in CD4+ splenic cells. Intranasal immunization with gB1s-NISV could elicit 90% (almost complete) protection against a heterologous lethal vaginal challenge with herpes simplex virus type 2. These data may have implications for the development of a mucosal vaccine against genital herpes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Future cancer therapies will be molecular cures. They will correct, block or destroy cancer cells by targeting molecular changes that lead to carcinogenesis. Destroying cancer cells can be done using oncolytic viruses. By blocking antibody mediated neutralization of oncolytic viruses, Herpes simplex virus type 1 glycoproteins E and I could be used in the adjuvant treatment of cancer for improving the chances of oncolytic viruses to kill cancer cells in vivo.

Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1-4 and WNV - New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI-TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus., (Copyright 2010 Elsevier Inc. All rights reserved.)

To develop a new type vaccine for porcine reproductive and respiratory syndrome (PRRS) prevention by using canine adenovirus 2(CAV-2) as vector, the Glycoprotein 5(GP5) gene from PRRSV strain JL was amplified by RT-PCR, and the expression cassette of GP5 was constructed using the human cytomegalovirus (HCMV) promoter and the simian virus 40 (SV40) early mRNA polyadenylation signal. The expression cassette of Glycoprotein 5 was cloned into the CAV-2 genome in which E3 region had been partly deleted, and the recombinant virus (CAV-2-GP5) was obtained by transfecting the recombinant CAV-2-GP5 genome into MDCK cells together with Lipofectamine 2000. Immunization trial in pigs with the recombinant virus CAV-2-GP5 showed that CAV-2-GP5 could stimulate a specific immune response to PRRSV. Immune response to the GP5 and PRRSV was confirmed by ELISA, neutralization test and lymphocyte proliferative responses, and western blotting confirmed expression of GP5 by the vector in cells. These results indicated that CAV-2 may serve as a vector for development of PRRSV vaccine in pigs, and the CAV-2-GP5 might be a candidate vaccine to be tested for preventing PRRSV infection.

We previously developed the bio-nanocapsule, which consists of hepatitis B virus envelope L proteins. The bio-nanocapsule can be used to deliver genes and drugs specifically to the human liver-derived tissues in xenograft models, presumably by utilizing the human liver-specific mechanism of hepatitis B virus infection. The hepatitis B virus tropism is highly restricted to humans and higher primates. Thus, to evaluate the in vivo therapeutic effects of forthcoming bio-nanocapsule-based medicines, it will be crucial to develop an animal model whose liver is susceptible to both bio-nanocapsule and hepatitis B virus. In the present study, we aimed to establish a bio-nanocapsule-susceptible animal model using transgenic rats expressing squamous cell carcinoma antigen-1 (SCCA1), which has been proposed to be a receptor for hepatitis B virus, interacting with the hepatitis B virus envelope protein and enhancing the cellular uptake of hepatitis B virus. We show that the recombinant SCCA1 protein interacts directly with bio-nanocapsule and inhibits its attachment to the cultured human liver-derived cells. Furthermore, we have established a transgenic rat that specifically expresses SCCA1 in the liver and also demonstrate that the amount of bio-nanocapsule accumulated in the liver is significantly increased by the SCCA1 expression. Histological analysis suggests that bio-nanocapsule is preferentially incorporated into the SCCA1-expressing hepatocytes but not into macrophages, such as Küppfer cells, nor into endothelial cells. Therefore, this animal model is expected to be useful for the development of bio-nanocapsule-based medicines.

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.

Previous studies conducted in our laboratory with Sindbis viral vectors in animal models demonstrated excellent in vivo targeting of tumor cells and significant reduction of metastatic implant size. To explore the influence of Sindbis strain on these factors, we constructed new plasmids from the wild-type Ar-339 Sindbis virus strain and compared their sequences. We found differences in the replicase and envelope proteins between JT, HRSP, and Ar-339 sequences. We made chimeras combining both strains and studied their efficiency in SCID mice bearing tumor xenograft using IVIS in vivo imaging techniques. We found that JT envelope proteins targeted tumors more efficiently than those of Ar-339, while the Ar-339 replicase showed increased efficacy in tumor reduction. To determine which residues are responsible for tumor targeting, we made mutants of Ar-339 E2 envelope protein and tested them by IVIS imaging in ES-2 tumor-bearing and tumor-free mice. The change of only one amino acid from E70 to K70 in Ar-339 E2 suppressed the ability to target metastatic tumor implants in mice. A K70 and V251 double E2 mutant did not reverse the loss of targeting capability. Only the mutant with JT E2 and Ar-339 helper targeted tumor, though with less intensity.

The guinea pig model of recurrent genital herpes simplex virus type 2 (HSV-2) infection was used to test the immunotherapeutic activity of a glycoprotein subunit vaccine. Vaccine formulation consisted of three recombinant herpes simplex virus (HSV) glycoproteins, namely gB1s, gD2t and gE1t, plus aluminium hydroxide [Al(OH)3)] adjuvant. One month after viral challenge, infected animals were therapeutically immunised by seven subcutaneous injections of a low dose of antigens with a weekly interval for the first five and a fortnightly interval for the last two administrations. Results showed that the treatment was highly effective in ameliorating the recidivist pathology of animals, suggesting that this kind of vaccine formulation and administration may be helpful for therapeutic intervention in humans affected by recurrent herpes infections.

A novel, general approach to chemical inactivation of retroviruses was used to produce inactivated simian immunodeficiency virus (SIV) particles with functional envelope glycoproteins. Inactivated virions of three different virus isolates (SIVmne E11S, SIVmac239, and SIVmac239 g4,5), prepared by treatment with 2,2'-dithiodipyridine (aldrithol-2, AT-2), were not detectably infectious, in vitro or in vivo. Immunization of pigtailed macaques with inactivated SIVmne E11S particles, without adjuvant, induced both humoral and cellular immune responses. Four of six animals immunized with the inactivated particles did not show measurable SIV RNA in plasma (<100 copy Eq/ml) following intravenous challenge with pathogenic, homologous virus (SIVmne E11S), compared to peak values of > or =10(6) copy Eq/ml in challenged SIV-naive control animals (p = 0.0001). Despite the absence of measurable viral RNA in plasma in these animals, culturable virus and viral DNA were initially detectable in blood and lymph node specimens; in contrast to control animals, SIV DNA could no longer be detected in PBMC by 10 weeks postchallenge in five of six SIV-immunized animals (p = 0.0001). However, vaccines did not resist a sequential rechallenge with the heterologous pathogenic virus SIVsm E660. AT-2-inactivated virus with functional envelope glycoproteins is a novel class of vaccine immunogen and was noninfectious, under conditions of rigorous in vivo challenge, and induced both binding and neutralizing antibody responses, along with cellular immune responses. Results suggest that immunization facilitated effective containment of pathogenic homologous challenge virus. With further optimization, AT-2-inactivated viral particles may be a useful class of immunogen in the development of a vaccine to prevent AIDS.

The coronavirus SARS-CoV is the primary cause of the life-threatening severe acute respiratory syndrome (SARS). With the aim of developing therapeutic agents, we have tested peptides derived from the membrane-proximal (HR2) and membrane-distal (HR1) heptad repeat region of the spike protein as inhibitors of SARS-CoV infection of Vero cells. It appeared that HR2 peptides, but not HR1 peptides, were inhibitory. Their efficacy was, however, significantly lower than that of corresponding HR2 peptides of the murine coronavirus mouse hepatitis virus (MHV) in inhibiting MHV infection. Biochemical and electron microscopical analyses showed that, when mixed, SARS-CoV HR1 and HR2 peptides assemble into a six-helix bundle consisting of HR1 as a central triple-stranded coiled coil in association with three HR2 alpha-helices oriented in an antiparallel manner. The stability of this complex, as measured by its resistance to heat dissociation, appeared to be much lower than that of the corresponding MHV complex, which may explain the different inhibitory potencies of the HR2 peptides. Analogous to other class I viral fusion proteins, the six-helix complex supposedly represents a postfusion conformation that is formed after insertion of the fusion peptide, proposed here for coronaviruses to be located immediately upstream of HR1, into the target membrane. The resulting close apposition of fusion peptide and spike transmembrane domain facilitates membrane fusion. The inhibitory potency of the SARS-CoV HR2-peptides provides an attractive basis for the development of a therapeutic drug for SARS.

Plasmid DNA synthesizing membrane-anchored or secretory Japanese encephalitis virus (JEV) envelope (E) protein and premembrane protein was delivered to mice by intramuscular injection or gene gun. Intramuscular plasmid immunization induced anti-E antibody responses similar to those associated with commercial JEV vaccine. The gene gun induced less antibody response. The 2 forms of the E protein induced similar antibody titers when administered by the same delivery mode. Both plasmids generated high titers of JEV-neutralizing antibodies, although the titers were lower than those induced by the vaccine. Intramuscular DNA immunization induced T helper 1 (Th1) immune responses, and the gene gun induced Th2 responses. Compared with secretory E protein, the membrane-anchored protein heavily skewed the immune response toward either Th1 or Th2, depending on the route of immunization. In an intracerebral JEV challenge model, plasmid-immunized mice had approximately 60% protection; this was not affected by the form of the E protein or by immunization route.

The need for an effective vaccine against HIV has prompted a refocusing of attention on mucosal immunity. More than 75% of all infections are acquired across a mucosal surface. It is therefore a prerequisite for a vaccine to target directly the mucosal tissues or indirectly the regional lymph nodes in order to prevent or control viral replication. Although mucosal immunization has induced responses at the genital or rectal surfaces, immune mechanisms alone have not been shown to be sufficient to contain infections in macaques. A growing body of evidence suggests that a dual mechanism may be required for effective mucosal protection, mediated by specific CD4 and CD8 T cell and antibody responses to the immunizing agents, plus innate antiviral factors and beta chemokines that down-regulate CCR5 coreceptors. Targeted iliac lymph node immunization with SIV gp 120 and p27 in alum prevents SIV infection or significantly decreases the viral load when immunized macaques were challenged with SIV by the rectal route. Indeed, in addition to specific immunity, including significant SIgA antibody secreting cells in the iliac lymph nodes, CD8-suppressor factor and the 3beta chemokines (RANTES, MIP-1alpha and MIP-1beta) are significantly associated with protection against rectal mucosal SIV infection.

A nucleic acid vaccine candidate against dengue-2 virus was constructed to express a truncated dengue-2 E glycoprotein without concomitant expression of prM. The truncated E protein was properly expressed even in the absence of prM. Mice inoculated intramuscularly with the recombinant plasmid containing 94% of the E gene did not respond with anti-dengue antibodies, cellular proliferation, or synthesis of cytokines by their lymphoid cells when stimulated with purified dengue-2 virus. However, protection was observed in 20% of the challenged mice immunized with this recombinant plasmid and the mice survived longer than the control group. The low percentage of protection might be explained by a weak activation of the immune system resulting from an imperfect secretion of E due to lack of the prM protein. This study corroborates with the hypothesis that prM is important for the processing of the E glycoprotein and should be incorporated on candidate vaccines engineered by recombinant DNA technology.

Classical swine fever virus causes significant mortality and morbidity in commercial piggeries in many countries in Europe and Asia. The protective antigen, gp55, is highly conformation-dependent and thus killed virus or bacterially produced proteins are not protective. This report demonstrates that DNA vaccination with the gene encoding gp55 can provide protective immunity with inoculation of two doses of 25 microg DNA or a single shot of 200 microg. Furthermore, the DNA can be delivered intramuscularly or by a simple spring-loaded needleless inoculator. In addition it is shown that inoculation of the DNA at a single site conveys the same level of immunity as division of the dose between two sites.

The ability of a genetically detoxified mutant of heat labile enterotoxin (LTK63) to act as a mucosal adjuvant following intranasal immunization with recombinant gD2 has previously been reported in mice [Ugozzoli M, O'Hagan DT, Ott GS. Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses. Immunol 1998;93:563-571.]. In the current studies, these observations were extended to the guinea pig model. Immunized guinea pigs were subsequently challenged intravaginally with HSV-2. Intranasal immunization with gD2 and LTK63 induced a significant reduction in disease severity and a reduction in mortality. However, only intramuscular immunization with a potent adjuvant (MF59) induced protection against the incidence of disease.

We constructed a recombinant baculovirus which produces a dengue (DEN)-2 virus envelope (E) protein containing a six-histidine (H6) tag in place of the last 100 amino acids at its C-terminus. The recombinant protein was purified from the supernatant of baculovirus-infected Spodoptera frugiperda insect cell cultures to apparent homogeneity by cation-chelation chromatography (TALON) in which the H6-tagged E-protein was eluted under non-denaturing conditions with 100 mM imidazole at pH 8.0. Mice vaccinated with the purified E mixed with aluminium hydroxide adjuvant showed an immune response of IgM and IgG1, IgG2a and IgG2b isotypes, and neutralizing antibodies, similar to that following immunization with purified inactivated DEN-2 virus. Moreover, mice that received the purified recombinant protein were significantly protected against 200 50% lethal dose of DEN-2 virus. This combination of affinity purified H6-tagged protein and adsorption onto a cationic carrier seems promising for the production of immunogenic particulate proteins, especially DEN protein for the four serotypes, and the development of a new generation of vaccines.

Liposomes were first proposed and tested as a drug delivery system 25 years ago. Since then, advances in our understanding of the fate and behaviour of liposomes at the cellular and subcellular level in vivo have allowed the rational design of constructs for use in the treatment and prevention of disease, both in experimental animals and clinically. The involvement of several liposome-based biotechnology companies in the early 1980s, paralleled by great leaps in liposome technology, has culminated in the design and licencing of formulations for the treatment of certain microbial infections and cancers, and the first liposome-based vaccine for use in humans.

Immunological analyses in this laboratory and others have suggested that a nonrecurrent HSV seropositive immune status is more closely correlated with a type 1 T helper cell (Th1) response characterized by elevated levels of interferon-gamma and IL2 rather than high titers of virus-specific antibodies. Effective intervention with an immunotherapeutic vaccine may require modulation of the regulatory network of T helper cells such that there is selective restimulation and expansion of the Th1 response. We have established a murine model for assessing the immunomodulatory capacity of an HSV glycoprotein subunit vaccine in animals with pre-existing herpes immunity. Animals were infected with varying doses of HSV1 and then administered glycoprotein D (gD) vaccine adjuvanted with aluminum phosphate at 3-week intervals. Observed changes in serological and cellular responses indicated that administration of subunit vaccine adjuvanted with aluminum phosphate could shift a dominant Th1 response, induced by sensitization with live HSV, towards a Th2 profile of activity. These data suggest that use of aluminum based adjuvants will not selectively stimulate Th1-associated responses and alternative adjuvants may be required for effective use of subunit vaccine in an immunotherapeutic indication in humans.

Immunotherapy of chronic viral diseases with vaccines is an important but unproven concept. We investigated the effect of a vaccine containing recombinant glycoprotein D (gD2) of herpes simplex virus type 2 (HSV-2) on the frequency of symptomatic outbreaks in patients with genital herpes. 98 patients with documented genital herpes who reported 4-14 recurrences per year were enrolled in a double-blind, placebo-controlled trial. Subjects received injections of either 100 micrograms gD2 in alum or alum alone (placebo) at 0 and 2 months, and recurrences were documented for 1 year. The vaccine was well tolerated. gD2 recipients reported fewer recurrences per month than placebo recipients (mean 0.42 [SE 0.05] vs 0.55 [0.05]; p = 0.055), had fewer virologically confirmed recurrences per month (0.18 [0.03] vs 0.28 [0.03]; p = 0.019), and had a lower median number of recurrences for the study year (4 [range 0-17] vs 6 [0-15]; p = 0.039). Neither genital recurrence nor the placebo vaccine had any discernible effect on HSV-2-specific antibody responses, but gD2 vaccine boosted neutralising antibodies to HSV-2 fourfold and gD2-specific titres sevenfold over baseline levels. These results inspire optimism about the potential use of vaccine for the treatment of chronic, recurring viral diseases.

Previous studies demonstrated that the adjuvant-free form of a fusion protein consisting of a truncated herpes simplex virus type 1 (HSV-1) glycoprotein D and human interleukin-2 (tgD-IL-2) elicited superior protective immunity in mice. In this study, the immunotherapeutic efficacy of tgD-IL-2 against vaginal HSV-2 infection was investigated using a guinea pig model. Footpad injections of tgD-IL-2 (12.5 micrograms/dose) after the onset of primary lesions strongly suppressed recurrence in the chronic phase of infection; consequently, the number of days with lesions was reduced 65%. Continuous medication with 100 mg/kg/day acyclovir for 5 days failed to suppress recurrent infection. In a UV radiation-induced recurrence model, prophylactic tgD-IL-2 significantly suppressed both duration and severity of disease. A single injection of tgD-IL-2 plus acyclovir produced an additive effect on the suppression of the disease in the acute phase. These results suggest that tgD-IL-2 is a promising immunotherapeutic agent against HSV-2 genital infections.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.

A monoclonal antibody (mAb), named 1.21, with complement-dependent neutralizing activity was produced against glycoprotein II (gII) of pseudorabies virus (PRV). By immunoaffinity chromatography using a mAB 1.21 column, gII was purified from Nonidet P40-lysates of PRV infected BHK21/13 cells. When mice and pigs were immunized with purified gII, complement-dependent virus-neutralizing antibodies were produced. The immunized animals survived potentially lethal challenge with PRV. These results indicate that an immunological response against gII plays an important role in the protection from PRV infection.

Reconstituted viral envelopes (RVEs) are formed by solubilizing intact virus in detergent and reassembling the envelope on removal of detergent. RVEs can be formed in the presence of agents that become encapsulated and can then be utilized in vitro and in vivo for drug delivery, cell destruction, transfer of membrane components, and as vectors for genetic engineering. The problems with biotechnological applications of RVEs and possible strategies for overcoming them are discussed in this article.

Subcutaneous injection of type 2 herpes simplex virus (HSV-2) (10(3) PFU) to newborn rabbits produced severe skin lesions and wide dissemination of the virus to various organs including the eye. Ocular lesions were characterized by retinal folds and choroiditis. HSV could be isolated from mononuclear cells (MNCs) of infected animal blood. Newborn rabbits treated with monoclonal antibody (MAb) against glycoprotein D (gD) of HSV on days 0, 2 and 4 postinfection had little or no skin lesions (0.0-2.3 mm) compared to controls (2.8-13.0 mm). In addition, the MAb treatment significantly suppressed dissemination of the virus to the eye (0% in MAb-treated vs 83% in control) and other organs and reduced the rate of chorioretinitis (0% in MAb-treated vs 50% in control). The treatment of HSV-infected MNCs with MAb resulted in 91-100% reduction in infectivity of the cells. The results suggest that anti-gD MAb protects newborn rabbits from HSV-2 eye infection by neutralizing the virus in skin and inactivating HSV-infected MNCs in blood.

Subunit pseudorabies vaccines that contained only purified glycoproteins of either of 2 strains of pseudorabies virus (PRV) were prepared and subsequently tested for safety and efficacy. The strains of virus used for vaccine production differed in at least 2 properties. One strain (Kojnok) was virulent for pigs and was believed to code for the entire complement of viral glycoproteins. The other (Kaplan) was a deletion mutant that was unable to code for structural viral glycoproteins gI and gp63. Purified glycoproteins were dispersed in an oil-in-water emulsion and were administered IM to pigs. Both vaccines were found to be safe and effective immunogens. Neither caused any local or general reactions, as verified by examination of the injection site (local safety) and by vaccination of pregnant sows in PRV-infected and noninfected herds. Sows vaccinated with the gI+ or gI- vaccine protected their pigs at levels of 93 and 92%, respectively, against a severe challenge exposure that killed 98% of pigs born from nonvaccinated sows. Vaccinated pigs were tested for active immunity by intranasal challenge exposure with the NIA 3 strain. Protection was quantitated by measuring the relative daily weight difference, expressed in percent per day, between vaccinated and control pigs during the first week after challenge exposure (delta G7); the estimated differences were 2.25 and 2.13% for gI+ and gI- vaccines, respectively. The absence of gI and gp63 did not affect the efficacy of this type of subunit glycoprotein vaccines.(ABSTRACT TRUNCATED AT 250 WORDS)

A chemically synthesized peptide consisting essentially of two separate regions (residues 141 to 158 and 200 to 213) of a virus coat protein (VP1) from the O1 Kaufbeuren strain of foot-and-mouth disease virus was prepared free of any carrier protein. It elicited high levels of neutralizing antibody and protected cattle against intradermolingual challenge by inoculation with infectious virus. Comparative evaluation of this peptide with a single-site peptide (residues 141 to 158) in guinea pigs suggests the importance of the VP1 carboxyl terminal residues in enhancing the protective response.

A recombinant, truncated HSV type 1 glycoprotein D secreted by Chinese hamster ovary cells (rgD1) was used to compare the ability of several adjuvants to stimulate protective immunity in guinea pigs. Adjuvants tested included CFA, aluminum hydroxide (alum), a lipophilic derivative of muramyl tripeptide (MTP-PE), and a muramyl dipeptide (MDP) covalently conjugated to rgD1. Animals were immunized three times with rgD1 plus the various adjuvants and antibody titers were determined by ELISA. Four weeks after the last immunization, the animals were challenged intravaginally with HSV type 2 and were monitored daily for clinical signs of disease, including frequency and severity of herpetic lesions, incidence of urinary retention, and mortality during the 14-day post-challenge observation period. Animals immunized in the foot-pad with rgD1 formulated with CFA showed the highest antibody titers. Animals immunized in the footpad with rgD1 using MTP-PE in a 4% squalene formulation, alum, or rgD1 conjugated to MDP showed mean antibody titers that were 57, 16, and 13% of the CFA titers, respectively. Immunization with rgD1 plus MTP-PE, alum, or rgD1-MDP conjugate by the i.m. route elicited lower antibody titers than the footpad route of immunization. Results of the viral challenge indicated that clinical symptoms of the groups immunized with rgD1 with CFA or MTP-PE as adjuvant were similar in magnitude and were markedly reduced compared with unimmunized control groups. Animals immunized with rgD1 combined with alum or rgD1-MDP conjugate showed clinical symptoms significantly more severe than the CFA or MTP-PE groups. The protective immunity observed after i.m. immunization of animals with rgD1 and MTP-PE was only slightly lower than animals immunized with the same Ag-adjuvant combination in the footpad. The results indicate that MTP-PE is an effective adjuvant for the recombinant herpes gD vaccine.

Treatment of previously infected guinea pigs with herpes simplex virus (HSV) glycoproteins reduces the frequency and severity of subsequent genital recurrences. An effective vaccine should also reduce episodes of viral shedding. In this study, HSV glycoproteins B and D treatment of animals experiencing recurrent genital herpes reduced the frequency of both clinical recurrences and cervicovaginal viral shedding. When virus was shed, however, the peak viral titer and duration of shedding was unaltered.

We investigated the possibility that herpes simplex virus (HSV) glycoproteins might provide potential immunotherapy for recurrent HSV disease by using the guinea pig model of genital HSV infection. In two experiments, after recovery from initial genital herpes, 58 animals were randomized either to receive a glycoprotein vaccine or to serve as controls. Both a mixture of HSV-2 glycoproteins derived from infected tissue cultures and a mixture of HSV-1 glycoproteins B and D, prepared by genetic engineering in mammalian cells, reduced the frequency (P less than .05) and severity (P less than .05) of subsequent herpetic recurrences. We conclude that recurrences of established herpesvirus disease may be altered by administering immunogenic viral proteins; thus, HSV glycoproteins may be useful as immunotherapeutic agents for controlling recurrent HSV infection in humans.