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PubMed2005

Identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants.

BACKGROUND: Recombination technology can be used to create live attenuated respiratory syncytial virus (RSV) vaccines that contain combinations of known attenuating mutations. METHODS: Two live attenuated, recombinantly derived RSV vaccine candidates, rA2cp248/404 Delta SH and rA2cp248/404/1030 Delta SH, were evaluated in 31 adults and in 95 children >/=6 months old. rA2cp248/404/1030 Delta SH was subsequently evaluated in 44 infants 1-2 months old. These vaccine candidates share 4 attenuating genetic elements and differ only in a missense mutation (1030) in the polymerase gene. RESULTS: Both vaccines were highly attenuated in adults and RSV-seropositive children and were well tolerated and immunogenic in RSV-seronegative children. Compared with that of rA2cp248/404 Delta SH, replication of rA2cp248/404/1030 Delta SH was restricted in RSV-seronegative children (mean peak titer, 10(4.3) vs. 10(2.5) plaque-forming units [pfu]/mL), indicating that the 1030 mutation had a potent attenuating effect. Although rA2cp248/404/1030 Delta SH was well tolerated in infants, only 44% of infants who received two 10(5.3)-pfu doses of vaccine had detectable antibody responses. However, replication after administration of the second dose was highly restricted, indicating that protective immunity was induced. At least 4 of 5 attenuating genetic elements were retained in recovered vaccine viruses. CONCLUSIONS: rA2cp248/404/1030 Delta SH is the first RSV vaccine candidate to be sufficiently attenuated in young infants. Additional studies are needed to determine whether rA2cp248/404/1030 Delta SH can induce protective immunity against wild-type RSV.

Adult↗
PubMed2000

QS-21 synergizes with recombinant interleukin-12 to create a potent adjuvant formulation for the fusion protein of respiratory syncytial virus.

In the murine model of respiratory syncytial virus (RSV) infection, recombinant interleukin-12 (rIL-12) was previously demonstrated to be most effective as an adjuvant for fusion (F) protein-based vaccines after adsorption to alum adjuvant. Alum, however, is a potent inducer of polarized T-cell responses and the type 2 phenotype. In addition, studies in human cancer patients demonstrated that rIL-12 was toxic when administered systemically at high doses. Because of these issues, we investigated whether the saponin QS-21 could facilitate a reduction in dose of rIL-12 when F protein was prepared in the absence of alum (F/rIL-12). The results demonstrated that a suboptimal dose (0.8 microg) of QS-21 enhanced the capacity of F/rIL-2 to elicit antigen-dependent killer cell precursors and complement-assisted neutralizing antibodies in spleens and sera respectively of BALB/c mice. The killer cell activity of mice vaccinated with F/rIL-12 (0.1 microg) plus 0.8 microg QS-21 was equivalent to that elicited after vaccination by experimental infection, or with an optimal dose of QS-21. The neutralizing titers generated by F/rIL-12 (0.01 to 1.0 microg) were also significantly elevated in the presence of 0.8 microg QS-21. Thus, rIL-12 and QS-21 together form a potent adjuvant for eliciting functional cell-mediated and humoral immune responses against F protein.

Adjuvants, Immunologic↗
PubMed2005

Recombinant Sendai virus as a novel vaccine candidate for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is among the most important and serious pediatric respiratory diseases, and yet after more than four decades of research an effective vaccine is still unavailable. This review examines the role of the immune response in reducing disease severity; considers the history of RSV vaccine development; and advocates the potential utility of Sendai virus (a murine paramyxovirus) as a xenogenic vaccine vector for the delivery of RSV antigens. The immunogenicity and protective efficacy of RSV-recombinant Sendai virus vectors constructed using reverse genetics is examined. RSV-recombinant Sendai virus is easy to grow (i.e., achieves extremely high titers in eggs), is easy to administer (intranasal drops), and elicits both B- and T-cell responses leading to protection from RSV challenge in a small-animal model. Unmodified Sendai virus is currently being studied in clinical trials as a vaccine for its closely related human cognate (human parainfluenza virus type 1). Sendai virus may prove an enormously valuable vaccine platform, permitting the delivery of recombinants targeting important pediatric respiratory pathogens, RSV chief among them.

Animals↗
PubMed2005

Respiratory syncytial virus infections: old challenges and new opportunities.

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia among children aged < 1 year. The majority of children hospitalized for RSV infection are younger than 6 months of age. RSV also causes repeated infections including severe lower respiratory tract disease, which may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. METHODS: Using the mouse model of RSV infection, this article examines the immunopathogenesis during acute and chronic phases of the disease. This model allows for measurement of basal enhanced pause, which reflects airway obstruction in the acute phase, and the response to methacholine challenge to assess airway hyperresponsiveness during the chronic phase. This article also summarizes some recent studies focusing on novel perspectives and strategies for treatment and prevention of RSV infections. RESULTS: Compared with the lungs of sham-inoculated control mice, mice inoculated with live RSV showed a persistent progression of the severity of pneumonia as determined by an increasing histopathologic score. Mucus production of RSV-infected mice in the acute phase illustrated increased periodic acid-Schiff-positive hypertrophic cells in central and peripheral airways. CONCLUSIONS: RSV-infected mice with persistent airway hyperresponsiveness exhibited the presence of abnormal chronic inflammatory changes and mucus overproduction, which likely contributed to long term airway disease induced by RSV infection. These findings provide a histologic correlation to the abnormal pulmonary responses documented by plethysmography. Current trials have demonstrated positive results in continuing to target different alternatives for a new RSV vaccine.

Adult↗
PubMed1996

Proposed three-dimensional model for the attachment protein G of respiratory syncytial virus.

Protein G of respiratory syncytial virus (RSV) is an envelope glycoprotein that is structurally very different from its counterparts (haemagglutinin-neuraminidase and haemagglutinin) in other paramyxoviruses. In this study, we put forward a model for this unique viral envelope protein. We propose that protein G of RSV contains several independently folding regions, with the ectodomain consisting of a conserved central hydrophobic region located between two polymeric mucin-like regions. The central conserved region is probably the only relatively fixed and folded part of the ectodomain of RSV-G. This central conserved region contains four conserved cysteine residues which can form two disulphide bridges. Analysis of the proteolytic digestion products of a peptide corresponding to the central conserved region showed that one of the three theoretically possible combinations of disulphide connections could be eliminated. The final disulphide bridge assignment was established by affinity measurements with peptide variants in which different disulphide connections were formed. Additionally, peptide binding studies were used to map the binding site, at the amino acid level, of a monoclonal antibody directed against the central conserved region. These studies indicated the level of surface exposure of the amino acid side-chains. The surface exposure agreed with the structural model. The proteolytic digestion, the peptide binding studies and the affinity measurements with structural peptide variants support a structural model with disulphide connections that correspond to a structural motif called a cystine noose. This model provides a structural explanation for the location and molecular details of important antigenic sites.

Amino Acid Sequence↗
PubMed1997

Analysis of bovine respiratory syncytial virus envelope glycoproteins in cell fusion.

To compare the requirements for respiratory syncytial virus (RSV)-mediated cell fusion, the fusion (F), attachment (G) and small hydrophobic (SH) glycoproteins of bovine RSV (BRSV), ovine RSV (ORSV) and human RSV (HRSV) were expressed individually or coexpressed in either homologous or heterologous combinations in HeLa cells, using the vaccinia virus-T7 polymerase transient expression system. Cell fusion was examined by a reporter gene activation assay. Although the expression of the F protein alone or coexpression of the F and G proteins or the F and SH proteins induced cell fusion, coexpression of F, G and SH proteins induced extensive cell fusion. Coexpression of various combinations of envelope glycoproteins of BRSV, ORSV and HRSV indicated that substitution of heterologous SH protein affects the effective fusigenic properties of the BRSV F protein far more than that obtained by substituting the heterologous G protein.

Animals↗
PubMed2001

Increased replication of respiratory syncytial virus (RSV) in pulmonary infiltrates is associated with enhanced histopathological disease in bonnet monkeys (Macaca radiata) pre-immunized with a formalin-inactivated RSV vaccine.

The pathology of respiratory syncytial virus (RSV) disease in bonnet monkeys parallels findings with human RSV disease. RSV-infected animals pre-immunized with a formalin-inactivated (FI) RSV vaccine develop inflammation in peribronchiolar, perivascular, interstitial and intra-alveolar sites with lung inflammation scores significantly higher than animals with a primary RSV infection and those pre-immunized with an FI-Vero cell control vaccine (P=0.05). Animals previously infected and re-exposed to RSV had significantly lower alveolar, interstitial and total lung inflammation scores than in primary infection (P=0.05). Immunization with two intra-muscular doses of 0.5 ml of the FI-RSV vaccine administered 21 days apart resulted in little serum-neutralizing and ELISA antibody, low levels of secretory IgA and a low lymphocyte proliferative response that was significantly lower than the response observed in animals that were previously infected with live RSV. Higher RSV virus titres were detected in the lungs and lung lavage fluid of monkeys immunized with the FI-RSV vaccine than in those with a primary infection (P=0.001). RSV was detected by in situ hybridization in pulmonary inflammatory infiltrates, where the single most abundant infiltrating cellular species was macrophages, so it may be these cells that support the enhanced virus replication that contributes to the enhanced pulmonary pathology of FI-RSV immunization.

Animals↗
PubMed2002

Virus-specific CTL responses induced by an H-2K(d)-restricted, motif-negative 15-mer peptide from the fusion protein of respiratory syncytial virus.

We describe 15-mer peptide P8:F92-106 from the F protein of respiratory syncytial virus (RSV) that can act as an MHC class I-restricted (H-2K(d)) epitope for RSV-specific CD8(+) CTL. This peptide is interesting because not only is it the first murine CTL epitope to be identified in the F protein but also because it does not contain a known allele-specific motif, as all 15 amino acids appear to be required for effective presentation to CTL. In in vitro MHC class I refolding experiments, peptide P8:F92-106 induced complex formation with H-2K(d) heavy chains and beta2-microglobulin. Immunization of BALB/c mice with P8:F92-106 resulted in the induction of peptide and RSV-specific CTL responses as well as peptide-specific proliferative responses. Following intranasal challenge with RSV, P8:F92-106-immunized mice showed a significant reduction in viral load in the lungs compared to that seen in unimmunized mice. Furthermore, passive transfer of purified CD8(+) lymphocytes into BALB/c scid mice prior to challenge with RSV also resulted in a reduction in the virus load in lungs of challenged mice. These results indicate the potential of synthetic peptide epitopes for the induction of protective immune responses against RSV infection.

Amino Acid Sequence↗
PubMed2003

Bovine parainfluenza virus type 3 (PIV3) expressing the respiratory syncytial virus (RSV) attachment and fusion proteins protects hamsters from challenge with human PIV3 and RSV.

Parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV) are the main causes of ubiquitous acute respiratory diseases of infancy and early childhood, causing 20-25 % of pneumonia and 45-50 % of bronchiolitis in hospitalized children. The primary goal of this study was to create an effective and safe RSV vaccine based on utilizing attenuated bovine PIV3 (bPIV3) as a virus vector backbone. bPIV3 had been evaluated in human clinical trials and was shown to be attenuated and immunogenic in children as young as 2 months of age. The ability of bPIV3 to function as a virus vaccine vector was explored further by introducing the RSV attachment (G) and fusion (F) genes into the bPIV3 RNA genome. The resulting virus, bPIV3/RSV(I), contained an insert of 2900 nt, comprising two translationally competent transcription units. Despite this increase in genetic material, the virus replicated to high titres in Vero cells. This recombinant virus expressed the RSV G and F proteins sufficiently to evoke a protective immune response in hamsters upon challenge with RSV or human PIV3 and to elicit RSV neutralizing and PIV3 haemagglutinin inhibition serum antibodies. In effect, a bivalent vaccine was produced that could protect vaccinees from RSV as well as PIV3. Such a vaccine would vastly reduce the respiratory disease burden, the associated hospitalization costs and, most importantly, decrease morbidity and mortality of infants, immunocompromised individuals and the elderly.

Animals↗
PubMed2006

Effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (RSV).

Little is known about the identities and roles of antigen-presenting cells upon exposure to antigens of respiratory syncytial virus (RSV). Here, we focused on elucidating the importance of alveolar macrophages in conferring protective immunity in mice administered a liposome-encapsulated recombinant fragment of the RSV G protein. Mice were depleted of alveolar macrophages by intranasal inoculation of liposome-encapsulated dichloromethylenediphosphonic acid (DMDP). Mice depleted of alveolar macrophages prior to immunization developed reduced levels of serum RSV-neutralizing antibody and showed dramatically impaired protection against RSV challenge. The severity of interstitial inflammation was also markedly reduced in macrophage-depleted mice. In conclusion, this study demonstrates a pivotal role for alveolar macrophages during exposure to liposome-encapsulated RSV antigen in initiating both protective and histopathological responses against RSV.

Animals↗
PubMed2000

Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen.

Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.

Animals↗
PubMed2005

Induction of mucosal B-cell memory by intranasal immunization of mice with respiratory syncytial virus.

The capacity of live or inactivated respiratory syncytial virus (RSV) to induce B-cell memory in respiratory-associated lymphoid tissues of mice was examined. Eight weeks after primary inoculation with either live or inactivated RSV, adult BALB/c mice were challenged with 4x10(5) PFU of RSV. Protection from viral shedding and mucosal production of RSV-specific antibodies were examined at various time points after challenge. We found that primary immunization with live, but not inactivated, RSV induced complete and durable protection upon challenge within the upper and lower respiratory tract. Also, primary immunization with live, but not inactivated, RSV enhanced the production of mucosal RSV-specific immunoglobulin A (IgA) upon challenge. Secondary mucosal IgA responses were characterized by (i) the early production of mucosal IgA by B cells that reside in organized nasal-associated lymphoid tissues, cervical lymph nodes, and bronchial lymph nodes, and (ii) the subsequent production of RSV-specific IgA by mucosal effector tissues, such as the tracheal lamina propria and lung. These findings suggest that primary infection of mice with live RSV might induce mucosal IgA-committed memory B cells. A greater understanding of the characteristics of RSA-specific mucosal memory B cells may facilitate the development of an RSV vaccine.

Administration, Intranasal↗
PubMed2005

Immune responses and disease enhancement during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is one of the commonest and most troublesome viruses of infancy. It causes most cases of bronchiolitis, which is associated with wheezing in later childhood. In primary infection, the peak of disease typically coincides with the development of specific T- and B-cell responses, which seem, in large part, to be responsible for disease. Animal models clearly show that a range of immune responses can enhance disease severity, particularly after vaccination with formalin-inactivated RSV. Prior immune sensitization leads to exuberant chemokine production, an excessive cellular influx, and an overabundance of cytokines during RSV challenge. Under different circumstances, specific mediators and T-cell subsets and antibody-antigen immune complex deposition are incriminated as major factors in disease. Animal models of immune enhancement permit a deep understanding of the role of specific immune responses in RSV disease, assist in vaccine design, and indicate which immunomodulatory therapy might be beneficial to children with bronchiolitis.

Adolescent↗
PubMed2000

Efficacy and safety studies of a recombinant chimeric respiratory syncytial virus FG glycoprotein vaccine in cotton rats.

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.

Animals↗
PubMed2001

Recombinant bovine/human parainfluenza virus type 3 (B/HPIV3) expressing the respiratory syncytial virus (RSV) G and F proteins can be used to achieve simultaneous mucosal immunization against RSV and HPIV3.

Recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3), a recombinant bovine PIV3 (rBPIV3) in which the F and HN genes were replaced with their HPIV3 counterparts, was used to express the major protective antigens of respiratory syncytial virus (RSV) in order to create a bivalent mucosal vaccine against RSV and HPIV3. The attenuation of rB/HPIV3 is provided by the host range restriction of the BPIV3 backbone in primates. RSV G and F open reading frames (ORFs) were placed under the control of PIV3 transcription signals and inserted individually into the rB/HPIV3 genome in the promoter-proximal position preceding the nucleocapsid protein gene. The recombinant PIV3 expressing the RSV G ORF (rB/HPIV3-G1) was not restricted in its replication in vitro, whereas the virus expressing the RSV F ORF (rB/HPIV3-F1) was eightfold restricted compared to its rB/HPIV3 parent. Both viruses replicated efficiently in the respiratory tract of hamsters, and each induced RSV serum antibody titers similar to those induced by RSV infection and anti-HPIV3 titers similar to those induced by HPIV3 infection. Immunization of hamsters with rB/HPIV3-G1, rB/HPIV3-F1, or a combination of both viruses resulted in a high level of resistance to challenge with RSV or HPIV3 28 days later. These results describe a vaccine strategy that obviates the technical challenges associated with a live attenuated RSV vaccine, providing, against the two leading viral agents of pediatric respiratory tract disease, a bivalent vaccine whose attenuation phenotype is based on the extensive host range sequence differences of BPIV3.

Animals↗
PubMed2002

Gamma interferon-dependent protection of the mouse upper respiratory tract following parenteral immunization with a respiratory syncytial virus G protein fragment.

The protective mechanisms induced in the mouse upper respiratory tract (URT) after intraperitoneal immunization with G2Na, a recombinant respiratory syncytial virus (RSV) G protein fragment (amino acid residues 130 to 230), were investigated. This protection was recently shown to be mediated by CD4(+) T cells and to be critically dependent on the cysteines and amino acids 193 and 194 (H. Plotnicky-Gilquin, A. Robert, L. Chevalet, J.-F. Haeuw, A. Beck, J.-Y. Bonnefoy, C. Brandt, C.-A. Siegrist, T. N. Nguyen, and U. F. Power, J. Virol. 74:3455-3463, 2000). On G2Na, we identified a domain (amino acid residues 182 to 198) responsible for the T-helper-cell activity. This region coincided with a peptide designed AICK (residues 184 to 198) which includes the previously identified murine and human T-helper-cell epitope on the native G protein (P. W. Tebbey, M. Hagen, and G. E. Hancock, J. Exp. Med. 188:1967-1972, 1998). Immunization with AICK, in alum or complete Freund's adjuvant, significantly reduced nasal RSV titers in normal BALB/c mice. However, although lung protection was induced, in contrast to the case with live RSV, neither AICK nor G2Na was able to prevent nasal infection in gamma interferon (IFN-gamma)-knockout mice. Anti-IFN-gamma neutralizing antibodies partially inhibited URT protection after administration to G2Na-immunized BALB/c mice. Furthermore, while purified CD4(+) T cells from BALB/c mice immunized with G2Na or AICK significantly reduced lung and nasal infection of naive recipient mice after adoptive transfer, the cells from IFN-gamma-knockout mice had no effect. Together, these results demonstrated for the first time that the T-helper-cell epitope of RSV G protein induces URT protection in mice after parenteral immunization through a Th1-type, IFN-gamma-dependent mechanism.

Adoptive Transfer↗
PubMed2002

Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.

Animals↗
PubMed2002

Mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups A and B and human parainfluenza virus type 3 by using a live cDNA-derived vaccine based on a host range-attenuated bovine parainfluenza virus type 3 vector backbone.

Reverse genetics was used to develop a two-component, trivalent live attenuated vaccine against human parainfluenza virus type 3 (HPIV3) and respiratory syncytial virus (RSV) subgroups A and B. The backbone for each of the two components of this vaccine was the attenuated recombinant bovine/human PIV3 (rB/HPIV3), a recombinant BPIV3 in which the bovine HN and F protective antigens are replaced by their HPIV3 counterparts (48). This chimera retains the well-characterized host range attenuation phenotype of BPIV3, which appears to be appropriate for immunization of young infants. The open reading frames (ORFs) for the G and F major protective antigens of RSV subgroup A and B were each placed under the control of PIV3 transcription signals and inserted individually or in homologous pairs as supernumerary genes in the promoter proximal position of rB/HPIV3. The level of replication of rB/HPIV3-RSV chimeric viruses in the respiratory tract of rhesus monkeys was similar to that of their parent virus rB/HPIV3, and each of the chimeras induced a robust immune response to both RSV and HPIV3. RSV-neutralizing antibody titers induced by rB/HPIV3-RSV chimeric viruses were equivalent to those induced by infection with wild-type RSV, and HPIV3-specific antibody responses were similar to, or slightly less than, after infection with the rB/HPIV3 vector itself. This study describes a novel vaccine strategy against RSV in which vaccine viruses with a common attenuated backbone, specifically rB/HPIV3 derivatives expressing the G and/or F major protective antigens of RSV subgroup A and of RSV subgroup B, are used to immunize by the intranasal route against RSV and HPIV3, which are the first and second most important viral agents of pediatric respiratory tract disease worldwide.

Animals↗