Up-to-date role of biologics in the management of respiratory syncytial virus.

INTRODUCTION: Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in young children and a substantial contributor to respiratory tract disease throughout life. Despite RSV being a high priority for vaccine development, there is currently no safe and effective vaccine available. There are many challenges to developing an RSV vaccine and there are limited antiviral drugs or biologics available for the management of infection. In this article, we review the antiviral treatments, vaccination strategies along with alternative therapies for RSV. AREAS COVERED: This review is a summary of the current antiviral and RSV vaccination approaches noting strategies and alternative therapies that may prevent or decrease the disease severity in RSV susceptible populations. EXPERT OPINION: This review discusses anti-RSV strategies given that no safe and efficacious vaccines are available, and therapeutic treatments are limited. Various biologicals that target for RSV are considered for disease intervention, as it is likely that it may be necessary to develop separate vaccines or therapeutics for each at-risk population.

Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing S gene from porcine epidemic diarrhea virus and VP7 gene from porcine rotavirus.

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).

Respiratory syncytial virus fusion nanoparticle vaccine immune responses target multiple neutralizing epitopes that contribute to protection against wild-type and palivizumab-resistant mutant virus challenge.

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in newborns, young children, elderly, and immune-compromised. The RSV fusion (F) glycoprotein is a major focus of vaccine development and the target of palivizumab (Synagis®) which is licensed as an immuno-prophylactic for use in newborn children at high risk of infection. However, clinical use of a narrowly targeted monoclonal antibodies leads to the generation of escape mutant strains that are fully resistant to neutralization by the antibody. Herein, we evaluated the RSV F nanoparticle vaccine (RSV F vaccine), produced as near-full-length, pre-fusogenic F trimers that form stable protein-detergent nanoparticles. The RSV F vaccine induces polyclonal antibodies that bind to antigenic site II as well as other epitopes known to be broadly neutralizing. Cotton rats immunized with the RSV F vaccine produced antibodies that were both neutralizing and protected against wild-type RSV infection, as well as against a palivizumab-resistant mutant virus. Use of aluminum phosphate adjuvant with the RSV F vaccine increased site II antibody avidity 100 to 1000-fold, which correlated with enhanced protection against challenge. The breadth of the vaccine-induced antibody response was demonstrated using competitive binding with monoclonal antibodies targeting antigenic sites Ø, II, IV, and VIII found on pre-fusion and post-fusion conformations of RSV F. In summary, we found the RSV F vaccine induced antibodies that bind to conserved epitopes including those defined as pre-fusion F specific; that use of adjuvant increased antibody avidity that correlated with enhanced protection in the cotton rat challenge model; and the polyclonal, high-avidity antibodies neutralized and protected against both wild-type and palivizumab-resistant mutant virus. These data support the ongoing clinical development of the aluminum phosphate adjuvanted RSV F nanoparticle vaccine.

A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 105 50% tissue culture infective dose(s) (TCID50)] D/OK (n = 6) or D/600 (n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine (n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 105 TCID50) with IDV D/OK (n = 6) or D/660 (n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.

Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency.

The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codon-optimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M282-90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8+ T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

Modeling maternal fetal RSV F vaccine induced antibody transfer in guinea pigs.

BACKGROUND: Protection of newborns and young infants against RSV disease via maternal immunization mediated by transplacental transfer of antibodies is under evaluation in third-trimester pregnant women with the RSV recombinant F nanoparticle vaccine (RSV F vaccine). Since the hemichorial placental architecture in guinea pigs and humans is similar, the guinea pig model was employed to assess RSV F vaccine immunogenicity in pregnant sows and to compare RSV-specific maternal antibody levels in their pups. METHODS: Thirty (30) presumptive pregnant guinea pigs were immunized on gestational day 25 and 46 with placebo (PBS), 30µg RSV F, or 30µg RSV F+400µg aluminum phosphate. Sera at delivery/birth (sows/pups) and 15 and 30 days post-partum (pups) were analyzed for the presence of anti-F IgG, palivizumab-competitive antibody (PCA) and RSV/A microneutralization (MN). RESULTS: The rates of pregnancy and stillbirth were similar between controls and vaccinees. The vaccine induced high levels of anti-F IgG, PCA and MN in sows, with the highest levels observed in adjuvanted vaccinees. Placental transfer to pups was proportional to the maternal antibody levels, with concentration effects observed for all immune measures. CONCLUSIONS: The RSV F vaccine was safe and immunogenic in pregnant guinea pigs and supported robust transplacental antibody transfer to their pups. Relative concentration of antibodies in the pups was observed even in the presence of high levels of maternal antibody. Guinea pigs may be an important safety and immunogenicity model for preclinical assessment of candidate vaccines for maternal immunization.

A recombinant vesicular stomatitis virus-based Lassa fever vaccine protects guinea pigs and macaques against challenge with geographically and genetically distinct Lassa viruses.

BACKGROUND: Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a "universal" LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models. METHODOLOGIES/PRINCIPLE FINDINGS: Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever.

Combined prophylactic and therapeutic cancer vaccine: enhancing CTL responses to HPV16 E2 using a chimeric VLP in HLA-A2 mice.

We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8(+) T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and anti-CD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8(+) T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogenous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c(+) and CD11c(+) CD40(+) double positive dendritic cells in mice that received immune-modulators, GM-CSF and anti-CD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.

Characterization of immune responses induced by intramuscular vaccination with DNA vaccines encoding measles virus hemagglutinin and/or fusion proteins.

Measles virus (MV) hemagglutinin (MV-H) and fusion (MV-F) proteins induce plaque reduction neutralizing (PRN) antibodies and cell-mediated immune responses that protect against clinical measles. DNA vaccines that encode MV-H and MV-F are being investigated as a new generation of measles vaccine to protect infants too young to receive currently licensed attenuated measles vaccines. However, it is unclear whether DNA vaccines encoding both MV-H and MV-F act synergistically to induce stronger immunity than immunization with plasmids encoding MV-H or MV-F alone. To address this question, we generated Sindbis virus-based pSINCP DNA vaccines that encode either MV-H or MV-F alone or bicistronic or fusion system vectors that encode both MV-H and MV-F (to mimic MV infection where both MV-H and MV-F proteins are expressed by the same mammalian cell). Mice immunized with DNA vaccine encoding MV-H alone developed significantly greater PRN titers than mice immunized with bicistronic constructs. Interestingly, the presence of MV-F in the bicistronic constructs stimulated serum MV-specific immunoglobulin G of reduced avidity. By contrast, mice immunized with bicistronic constructs induced equivalent or higher levels of MV-specific gamma interferon responses than mice immunized with DNA vaccine encoding MV-H alone. These data will help guide the design of DNA-based MV vaccines to be used early in life in a heterologous prime-boost strategy.

[Enhancement of cellular immune response to DNA vaccine encoding hepatitis C virus core and envelope 2 fusion antigen by murine Fms-like tyrosine kinase 3 ligand].

Hepatitis C virus (HCV) is an important human pathogen that causes chronic liver disease worldwide. It is desirable to develop vaccines to prevent HCV infection, or at least to prevent progression to chronicity. We once constructed an optimized hepatitis C virus core and envelope 2 fusion antigen DNA vaccine, which could induce humoral and cellular immune responses against HCV core and E2 protein in BALB/c mice efficiently. Flt3 (Fms-like tyrosine kinase 3) -ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells, particularly dendritic cells. We reasoned that a DNA vaccine coexpressing the antigen and FL may activate immune responses more effectually. In this study, The influence of FL on this HCV DNA vaccine was evaluated. The cDNA encoding signal peptide and extracellular domain of murine FL was inserted into the plasmid pST-CE2t, and the resulting plasmid pST-CE2t/FL was transfected into COS7 cells. The HCV core and E2 protein were detected by Western blotting, and the soluble murine FL was detected by ELISA. Eight-week-old female BALB/c mice were inoculated intramuscularly with 100 microg pST-CE2t, pST-CE2t/FL or mock vector, respectively, and boosted at the same dosage 3 weeks later. Anti-HCV core and E2 total IgG and isotypes were measured at weeks 1,3,5,7. Splenocyte proliferative response to recombinant HCV core and E2 protrein were detected at week 7. SP2/0 cells expressing HCV core protein were used as target cells for the detection of cytotoxic T lymphocyte (CTL) response. Western blot analysis showed that a protein band with molecular weight about 70 kD from lysate of COS7 cells transfected with plasmid pST-CE2t/FL could be detected by anti-HCV core or E2 monoclonal antibodies, which indicated that pST-CE2t could express glucosylated HCV core and E2 fusion protein. Murine FL could be detected in the culture supernatant of COS7 cells transfected with pST-CE2t/FL. Plasmid pST-CE2t immunized mice developed higher anti-HCV core and E2 IgG seroconversion rates and titers than pST-CE2t/FL group did at different various times, but the IgG2a/IgG1 ratio of anti-HCV E2 protein in pST-CE2t/FL group is much higher than pST-CE2t group. Splenocytes from pST-CE2t or pST-CE2t/FL immunized mice could proliferate with stimulation of HCV core or E2 protein in vitro, although pST-CE2t/FL group showed much stronger response. Splenocytes from mice immunized with pST-CE2t/FL induced 79.03% +/- 9.95% of target cell lysis at the effector/target ratio of 100:1, which was significantly greater than the lysis (62.2% +/- 8.62%) observed in mice immunized with pST-CE2t. Our data demonstrated that the incorporation of FL can preferentially enhance the cellular response to this HCV fusion antigen DNA vaccine. In contrast, HCV specific antibodies were inhibited by FL in vaccinated mice. More and more data supports that recovery from acute HCV infection may depend upon the generation of broad-based cellular immune responses to viral proteins. So, FL may be of potential value as an adjuvant in the development of DNA-based immunization for prophylactic and therapeutic vaccine against HCV infection.

Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline.

Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T: residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6: residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment. However, helix association is accompanied by loss of overall helical content. Thermal-dependence studies show that the unfolding of self-associated alpha-helices is significantly more pronounced than the unfolding of isolated alpha-helices. Circular dichroism data, together with tryptic limited proteolysis, suggest the presence of a charged amino acid within the hydrophobic core. This study should provide a basis for engineering more effective immunogenic peptides against the measles virus by increasing the stability of the TT-NP6 peptide.

Evaluation of subunit vaccines against feline immunodeficiency virus infection.

Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase (K-SU3) or glutathione-S-transferase (G-SU3). Quantitative and qualitative differences in the humoral immune response were observed with three adjuvants of which Quil A was the best in terms of total and virus neutralizing antibody. Notwithstanding the responses induced, 19 of 20 immunized cats did not resist challenge and became infected. To determine whether priming with a live viral vector would confer protection, cats were inoculated oronasally and subcutaneously with a feline herpesvirus (FHV) mutant expressing the FIV env gene; two booster immunizations followed using the K-SU3 product in either Quil A or a mineral oill Al(OH)3 adjuvant. FIV-specific antibody responses were only weak, and the vaccinates did not withstand challenge with a low dose of homologous virus.

Reversible conformational changes and fusion activity of rabies virus glycoprotein.

In an attempt to understand the implication of the rabies virus glycoprotein (G) in the first steps of the viral cycle, we studied the pH dependence of virus-induced fusion and hemagglutination, as well as modifications of the structure and properties of the viral glycoprotein following pH acidification. Our results suggest that the G protein adopts at least three distinct configurations, each associated with different properties. At neutral pH, G did not fuse membranes or hemagglutinate erythrocytes. It was insensitive to digestion with bromelain and trypsin. At pH 6.4, the glycoprotein became sensitive to proteases. Hemagglutination was at its maximum and then sharply decreased with the pH. No fusion was detected. Aggregation of virus was also observed. The third configuration, at below pH 6.1, was associated with the appearance of fusion. Some neutralizing monoclonal antibodies were able to differentiate these three configurations. Preincubation of the virus at below pH 6 inhibited fusion, but this inhibition, like the structural modifications of the glycoprotein, was reversible when G was reincubated at neutral pH.

Evaluation of the immunogenicity and protective efficacy of a candidate parainfluenza virus type 3 subunit vaccine in cotton rats.

A parainfluenza virus type 3 (PIV3) subunit vaccine consisting of detergent-solubilized, affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in cotton rats for immunogenicity, short-term effects on virus-induced immunopathology and protective efficacy. Groups of animals were immunized twice, 4 weeks apart, with graded doses of vaccine administered either alone or with aluminium phosphate (AlPO4). The minimum immunogenic dose of vaccine was 0.1 microgram HN and F when the vaccine was given alone and 0.01 microgram when the vaccine was administered with AlPO4 adjuvant. Antibody responses in animals immunized with 1 microgram HN and F mixed with adjuvant were similar to those in control animals infected with live PIV3 intranasally. Pulmonary and nasal wash PIV3 titres generally were inversely correlated with serum antibody levels. Virus titres were significantly reduced in all groups of animals immunized with greater than or equal to 0.1 microgram HN and F compared with control animals immunized with vehicle only. Four days after virus challenge, there was no evidence of enhanced histopathology in lung sections from animals immunized with the candidate vaccine.