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Effects of dual vaccination for bovine respiratory syncytial virus and Haemophilus somnus on immune responses.

Bovine respiratory syncytial virus (BRSV) and Haemophilus somnus (H. somnus) co-infect to form a polymicrobial respiratory disease in calves. Both BRSV and H. somnus vaccinations have independently been shown to sometimes induce adverse IgE mediated responses. We hypothesized that combining these disease agents in vaccination would induce cytokine shifts resulting in greater IgE production and enhanced disease. Concurrent vaccination with subsequent infection with one or both pathogens in calves was conducted to evaluate the isotypic antibody responses, disease severity and cytokine response. BRSV-specific serum IgE levels were elevated for the most clinically diseased calves, while no difference was detected in the IgE levels to H. somnus among groups. The IFN-gamma message and H. somnus-specific IgG2 antibodies were significantly elevated in calves with the lowest clinical scores. Vaccination preferentially stimulated higher levels of IgG1 antibodies to BRSV, but in contrast higher levels of IgG2 antibodies to H. somnus. Concurrent vaccination induced IgE antibodies to BRSV, which were directly correlated with disease severity whereas vaccine induced IgG2 antibodies to H. somnus were inversely correlated with disease severity.

Animals↗

Role of respiratory syncytial virus in acute otitis media: implications for vaccine development.

We summarize herein the results of various virologic studies of acute otitis media (AOM) conducted at our site over a 10-year period. Among 566 children with AOM, respiratory syncytial virus (RSV) was the most common virus identified in either middle ear fluid or nasal wash; it was found in 16% of all children and 38% of virus-positive children. Seventy-one percent of the children with RSV were 1 year of age or older, which was significantly older than all other viruses combined (P=0.045). RSV infection was associated with the common bacterial pathogens causing AOM. Past efforts to develop vaccines for RSV have emphasized prevention of lower respiratory tract infection in infants, which is a more serious problem but less common than AOM. Our results suggest that RSV vaccines that work only against infection in older children may have value in preventing AOM, the most common pediatric disease.

Acute Disease↗

Formulation with CpG oligodeoxynucleotides increases cellular immunity and protection induced by vaccination of calves with formalin-inactivated bovine respiratory syncytial virus.

Vaccination of calves with formalin-inactivated bovine respiratory syncytial virus (FI-BRSV) induces low levels of cellular immunity that may not be protective. Since inactivated and subunit vaccines formulated with CpG oligodeoxynucleotides (ODNs) have been shown to induce cellular immune responses, we studied the ability of a FI-BRSV vaccine formulated with CpG ODN to elicit cellular immunity against BRSV. Neonatal calves were immunized with FI-BRSV, FI-BRSV formulated with CpG ODN or medium and challenged with BRSV after two immunizations. Calves vaccinated with FI-BRSV formulated with CpG ODN developed increased numbers of IFN-gamma secreting cells in the peripheral blood and broncho-tracheal lymph nodes and enhanced BRSV-specific serum IgG2 in comparison to FI-BRSV immunized animals. Calves that received the FI-BRSV vaccine formulated with CpG ODN also experienced a reduction in the amount of BRSV in the lung tissue. Based on these observations, CpG ODN appears to be a suitable candidate adjuvant for inactivated BRSV vaccines.

Adjuvants, Immunologic↗

Cytotoxic T lymphocyte activity and cytokine expression in calves vaccinated with formalin-inactivated bovine respiratory syncytial virus prior to challenge.

The development of effective, safe vaccines for human and bovine respiratory syncytial virus (RSV) has been problematic. Inactivated RSV vaccines are of variable efficacy; poor efficacy may be related to induction of ineffective cell-mediated immunity (CMI). To characterize CMI in calves vaccinated with formalin inactivated (FI) BRSV, 11 calves were vaccinated twice with FI-BRSV (n=5) or mock vaccine (n=6) at a 2 week interval and challenged 1 month later. Prior to challenge a cannula was placed in the efferent lymphatic of the caudal mediastinal lymph node of each calf; lymph derived lymphocytes (LDL) were collected for analysis of CMI. Cytotoxic T lymphocyte (CTL) activity by LDL and/or peripheral blood mononuclear cells (PBMC) was measured by 51Cr release on days 5, 7, 9, and 10 post-challenge. Messenger RNA for interferon gamma (IFN-gamma), interleukin 2 (IL-2) and IL-4 was measured on days 0-10 by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA of LDL. BRSV-specific IFN-gamma production by PBMC was measured on days 0 and 10 by ELISA. Clinical signs and postmortem changes following challenge were evaluated. There was no difference between groups in clinical signs, postmortem changes, CTL activity, cytokine message expression, or IFN-gamma production. For both groups, percentage lysis by CTL peaked on days 7-10 and ranged from 11 to 25%. Failure of vaccination to prevent disease following challenge was likely associated with failure to prime for improved CMI responses.

Animals↗

Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR.

In the murine model for respiratory syncytial virus (RSV) infection, cytokine patterns induced by vaccinations with either killed (i.e. formalin-inactivated, alum-precipitated) virus (KV) or live virus (LV) have been shown to influence disease expression. To determine the mRNA expression of the cytokines IL-4 and IFN-gamma in BALB/c mice challenged with RSV, a real-time quantitative reverse-transcriptase PCR assay was developed. This assay uses 5'-exonuclease fluorogenic probes and is performed on the ABI PRISM 7700 Sequence Detector System (TaqMan). The relative quantitative levels of mRNA for IL-4 and IFN-gamma were compared with those measured by an RNase protection assay (RPA) and an enzyme immunoassay (EIA), which are methods used to measure the levels of mRNA and protein, respectively. Results obtained by the TaqMan assay showed that mice primed with KV induces increased IL-4 mRNA production while LV induces increased IFN-gamma mRNA, which is in agreement with conventional methods. IL-4 and IFN-gamma relative quantities obtained from TaqMan were highly correlated to those determined by RPA (r=0.96 for IFN-gamma, P<0.01) and EIA (r=0.90 for IL-4 and r=0.75 for IFN-gamma, P<0.01). Assay reproducibility was examined by testing a same sample in triplicate at three experiments. Minimal deviation values were observed in both intra- and inter-assays. TaqMan, which is rapid, sensitive and reproducible, provides an alternative tool for the quantitative analysis of cytokine mRNA expression in the murine model of RSV immunopathogenesis.

Animals↗

Enhanced immune protection by a liposome-encapsulated recombinant respiratory syncytial virus (RSV) vaccine using immunogenic lipids from Deinococcus radiodurans.

The radiation-resistant bacterium, Deinococcus radiodurans contains a variety of phospho-, glyco- and phosphoglycolipids, the structures of which appear to be largely unique in nature. We show here that such lipids are immunogenic when administered as liposomes intranasally in mice, as evidenced by the induction of serum antibodies which recognize D. radiodurans lipids but not other lipids by thin layer chromatographic immunostaining. By modifying a liposomal vaccine against respiratory syncytial virus (RSV) we find that vaccine efficacy is markedly enhanced by the inclusion of lipids isolated from D. radiodurans. Using dioleoylphosphatidylcholine (DOPC) or D. radiodurans lipids, liposomes were prepared which encapsulated a soluble fragment of the RSV G protein (G(128-188)) fused with a portion of the bacterial thioredoxin (Trx) protein. Mice immunized intranasally with D. radiodurans liposomes showed markedly greater protection against RSV challenge over mice immunized with DOPC liposomes. Enhanced vaccine efficacy was achieved using liposomes prepared from either whole D. radiodurans lipids or from a single isolated phosphoglycolipid previously identified as alpha-galactosylphosphatidylglyceroylalkylamine (lipid 7). Mice immunized and protected against RSV challenge were free of pulmonary eosinophilic infiltration, an undesirable consequence of many RSV vaccines. The results provide further support for liposome-based vaccines for RSV and underline the importance of lipid composition in liposome formulations.

Animals↗

A single amino acid substitution in a recombinant G protein vaccine drastically curtails protective immunity against respiratory syncytial virus (RSV).

Recent studies have indicated a dominant T cell epitope located approximately between amino acids 184 and 203 on the respiratory syncytial virus (RSV) G protein. Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128-229) of the RSV G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids on vaccine efficacy. By changing two amino acids Arg 188 and Lys 192 to alanine, the ability of the Trx-G 128-229 fusion protein to protect mice against RSV challenge was virtually abolished. Mice immunized with the double mutant protein showed low levels of neutralizing antibodies and no pulmonary eosinophilic infiltrate, in contrast to that observed in mice immunized with wild type protein prior to RSV challenge. While less effective than the double mutant, mutation of either Arg 188 or Lys 192 to Ala drastically impaired the ability of immunized Trx-G 128-229 to induce neutralizing antibodies and to elicit pulmonary eosinophilia associated with RSV challenge. Despite low levels of virus-neutralizing antibodies, G protein-specific antibodies were detected by Western blotting in the sera from mice immunized with either of the single mutants (Arg 188 or Lys 192) but not the double mutant. Finally, immunization of mice with truncated forms of the Trx-G protein, showed partial protection against RSV challenge with Trx-G 128-188 but not with Trx-G 189-229. Taken together, the results indicate an important role for Arg 188 and Lys 192 in the induction of protective immunity and priming for eosinophilia against RSV. Furthermore, while the dominant protective linear epitope on the RSV G protein requires an intact sequence around Arg 188, there are additional, but less potent, protective epitopes upstream of Arg 188.

Amino Acid Substitution↗

CD40 ligand (CD154) improves the durability of respiratory syncytial virus DNA vaccination in BALB/c mice.

Respiratory syncytial virus (RSV) infection is the single most important cause of serious acute respiratory illness in children <1 year of age worldwide, and is associated with life-threatening pneumonia or bronchiolitis in the elderly. Current vaccine strategies include live, attenuated virus, subunit and DNA vaccines, however, none have been sufficiently safe, or shown to induce satisfactory long-term immunity, thus immune modulators are being considered to enhance the effectiveness of RSV vaccines. In this study, we examine CD40 ligand (CD40L) as an immune modulator to enhance the durability of DNA vaccines encoding RSV F and/or G glycoproteins in BALB/c mice. The addition of CD40L to DNA vaccines encoding the F glycoprotein enhanced virus clearance and some aspects of the immune response to RSV challenge, suggesting that CD40L may enhance the durability of RSV DNA vaccines.

Animals↗

The immunogenicity, protective efficacy and safety of BBG2Na, a subunit respiratory syncytial virus (RSV) vaccine candidate, against RSV-B.

Respiratory syncytial virus (RSV) is divided into subgroups A and B, based primarily on variation within the G glycoprotein. A safe vaccine that protects against both would be the ideal. BBG2Na is a recombinant subunit RSV vaccine candidate derived in part from the G protein of RSV-A. Interestingly, BBG2Na formulated in alum protected against RSV-B challenge at early time points following vaccination in mice. Over 6 months, however, BBG2Na-induced immunogenicity and protective efficacy progressively diminished, such that few animals were considered protected at the end. To study the safety of BBG2Na relative to RSV-B challenge, we established a novel enhanced immunopathology mouse model. We confirmed that RSV-B challenge of formalin-inactivated RSV-A (FI-RSV-A)-immunized BALB/c mice results in enhanced pulmonary pathology. Therefore, this phenomenon is neither subgroup-specific nor dependent on a previously incriminated Th epitope in the RSV-A G protein. In stark contrast, BBG2Na did not induce any signs of enhanced pulmonary pathology. In conclusion, our data indicate that BBG2Na, formulated in alum, induces safe and protective immune responses against RSV-B challenge in mice. However, the duration of protective immunity will probably be insufficient to prevent RSV-B infection for the duration of the RSV epidemic season.

Animals↗

Complete protection of mice from respiratory syncytial virus infection following mucosal delivery of synthetic peptide vaccines.

We have previously shown that intraperitoneal immunization of BALB/c mice with the 14 amino-acid long synthetic peptides G/174-187 and BG/174-187, representing the region 174-187 of the G-glycoprotein from human (H) and bovine (B) respiratory syncytial virus (RSV), respectively, completely protects animals from infection with the corresponding virus. A current goal in vaccine development being the delivery of noninvasive protective antigens via mucosal surfaces, we have evaluated the immunogenicity and protective efficacy of the two peptides when administered to mice by the intranasal (i.n.) route in the presence or absence of the cholera toxin (CT) as a mucosal adjuvant. The two peptides given alone induced the production of RSV-specific circulating IgG, as revealed by ELISA titers of immune sera. When the peptides were administered intranasally with CT, the higher IgG antibody titer which was induced was within the same order of magnitude as that obtained following i.n. immunization with live RSV or intraperitoneal injection with the peptides, thus demonstrating the stimulatory effect of the CT adjuvant. Moreover, although the peptides fail to induce a detectable level of secretory IgA, all animals immunized i.n. with peptide BG/174-187 (plus or minus CT) and all those immunized with peptide G/174-187 mixed with CT were completely resistant to infection by the corresponding virus. To our knowledge, this is the first study reporting that complete protection against a natural pathogen can be elicited by mucosally delivered synthetic peptides. This supports the usefulness of synthetic peptides in prophylactic vaccination.

Administration, Intranasal↗

Preventive and therapeutic strategies for respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) remains an important cause of pneumonitis in infants and in the elderly and immunosuppressed. Passive immunoprophylaxis of high-risk infants with the humanised monoclonal antibody palivizumab reduces RSV-related hospitalisation and admission to intensive care units by about 50%, although optimal and cost-effective use of this agent remains undefined. The development of an RSV vaccine is the focus of much research and recent advances with live attenuated vaccines are promising--several agents are in clinical trials. The value of the only therapeutic agent available to treat established infection, the antiviral ribavirin, is now seriously doubted and the agent is little used. New RSV chemotherapeutics are just entering clinical trials.

Animals↗

Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response.

Respiratory syncytial virus (RSV) is one of the most important pathogens of infancy and early childhood. Here a fruit-based edible subunit vaccine against RSV was developed by expressing the RSV fusion (F) protein gene in transgenic tomato plants. The F-gene was expressed in ripening tomato fruit under the control of the fruit-specific E8 promoter. Oral immunization of mice with ripe transgenic tomato fruits led to the induction of both serum and mucosal RSV-F specific antibodies. The ratio of immunoglobulin subclasses produced in response to immunization suggested that a type 1 T-helper cell immune response was preferentially induced. Serum antibodies showed an increased titer when the immunized mice were exposed to inactivated RSV antigen.

Administration, Oral↗

Structures of respiratory syncytial virus G bound to broadly reactive antibodies provide insights into vaccine design.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in infants and older adults. The attachment glycoprotein (RSV G) binds to the chemokine receptor CX3CR1 to promote viral entry and modulate host immunity. Antibodies against RSV G are a known correlate of protection. Previously, several broadly reactive, high-affinity anti-RSV G human monoclonal antibodies were isolated from RSV-exposed individuals and were shown to be protective in vitro and in vivo. Here, we determined the structures of three of these antibodies in complex with RSV G and defined distinct conformational epitopes comprised of highly conserved RSV G residues. Binding competition and structural studies demonstrated that this highly conserved region displays two non-overlapping antigenic sites. Analyses of anti-RSV G antibody sequences reveal that antigenic site flexibility may promote the elicitation of diverse antibody germlines. Together, these findings provide a foundation for next-generation RSV prophylactics, and they expand concepts in vaccine design for the elicitation of germline lineage-diverse, broadly reactive, high-affinity antibodies.

Humans↗

Respiratory syncytial virus infections in children and adults.

Respiratory syncytial virus is the leading cause of hospital admission for lower respiratory tract infection in young children and appears to be responsible for a significant burden of disease in adults, particularly the elderly and the immunocompromised. In this review, we describe the epidemiology, diagnosis and clinical manifestations of infection attributed to this virus. We also consider current therapeutic and prophylactic options and appraise strategies for vaccination that are in clinical trials.

Adult↗

Self-replicative RNA vaccines elicit protection against influenza A virus, respiratory syncytial virus, and a tickborne encephalitis virus.

In genetic vaccination, recipients are immunized with antigen-encoding nucleic acid, usually DNA. This study addressed the possibility of using the recombinant alpha virus RNA molecule, which replicates in the cytoplasm of transfected cells, as a novel approach for genetic vaccination. Mice were immunized with recombinant Semliki Forest virus RNA-encoding envelope proteins from one of 3 viruses: influenza A virus, a tickborne flavivirus (louping ill virus), or respiratory syncytial virus (RSV). Serologic analyses showed that antigen-specific antibody responses were elicited. IgG isotyping indicated that predominantly Th1 type immune responses were induced after immunization with RSV F protein-encoding RNA, which is relevant for protection against RSV infection. Challenge infection showed that RNA immunization had elicited significant levels of protection against the 3 model virus diseases.

Animals↗

Antibody-dependent enhancement, a possible mechanism in augmented pulmonary disease of respiratory syncytial virus in the Bonnet monkey model.

Bonnet monkeys develop an enhanced disease after immunization with the formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine that is characterized by increased viral replication in perivascular sites of the lung. These sites contain many mononuclear cells, which are known to be permissive for RSV replication. To test the hypothesis that FI-RSV vaccine stimulates the production of enhancing antibodies that serve to increase the replication of RSV in macrophages, in vitro studies were done. Antibody-dependent enhancement was observed in animals immunized with FI-RSV but not in control animals with primary and tertiary infections or those immunized with FI-Vero cell culture. In the presence of serum samples from animals immunized with FI-RSV, an increased number of U937 cells was infected. The enhancement index correlated positively with the pathologic scores of the FI-RSV-vaccinated monkeys.

Animals↗

Progress in the development of respiratory syncytial virus and parainfluenza virus vaccines.

Respiratory syncytial virus (RSV) and human parainfluenza viruses (hPIVs) are leading causes of viral lower respiratory tract illness in children and in high-risk adult populations. Despite decades of research, licensed vaccines for RSV and hPIVs do not exist. Recently, however, genetically engineered live attenuated RSV and hPIV candidate vaccines have been generated, several of which are already being evaluated in clinical trials. Recombinant technology allows candidate vaccines to be "fine-tuned" in response to clinical data, which should hasten the development of vaccines against these important respiratory pathogens.

Adult↗

Anti-G protein antibody responses to respiratory syncytial virus infection or vaccination are associated with inhibition of G protein CX3C-CX3CR1 binding and leukocyte chemotaxis.

Respiratory syncytial virus (RSV) is an important cause of severe lower respiratory tract illness in infants and the elderly. Presently, no safe and efficacious RSV vaccine exists; however, advances in our understanding of immunity and the pathogenesis of disease associated with RSV infection may lead to new vaccine strategies. RSV G protein contains a CX3C chemokine motif that interacts with the CX3CR1 chemokine receptor and modifies the activities of fractalkine. In the present study, we show that anti-RSV G protein antibody responses after recent RSV infection or vaccination are associated with inhibition of RSV G protein CX3C-CX3CR1 interaction and RSV G protein-mediated leukocyte chemotaxis.

Adult↗