Antibody recognition of the Pneumovirus fusion protein trimer interface.

Human metapneumovirus (hMPV) is a leading cause of viral respiratory infection in children, and can cause severe lower respiratory tract infection in infants, the elderly, and immunocompromised patients. However, there remain no licensed vaccines or specific treatments for hMPV infection. Although the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological properties of hMPV F remain poorly understood. To further define the humoral immune response to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and MPV465. Both mAbs are neutralizing in vitro and were determined to target a unique antigenic site using competitive biolayer interferometry. We determined both MPV458 and MPV465 have higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV F protein, suggesting significant breathing of the hMPV F protein must occur for host immune recognition of the novel epitope. In addition, significant glycan interactions were observed with a somatically mutated light chain framework residue. The data presented identifies a novel epitope on the hMPV F protein for epitope-based vaccine design, and illustrates a new mechanism for human antibody neutralization of viral glycoproteins.

A gammaherpesvirus licenses CD8 T cells to protect the host from pneumovirus-induced immunopathologies.

Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein-Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.

Antibody Epitopes of Pneumovirus Fusion Proteins.

The pneumoviruses respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two widespread human pathogens that can cause severe disease in the young, the elderly, and the immunocompromised. Despite the discovery of RSV over 60 years ago, and hMPV nearly 20 years ago, there are no approved vaccines for either virus. Antibody-mediated immunity is critical for protection from RSV and hMPV, and, until recently, knowledge of the antibody epitopes on the surface glycoproteins of RSV and hMPV was very limited. However, recent breakthroughs in the recombinant expression and stabilization of pneumovirus fusion proteins have facilitated in-depth characterization of antibody responses and structural epitopes, and have provided an enormous diversity of new monoclonal antibody candidates for therapeutic development. These new data have primarily focused on the RSV F protein, and have led to a wealth of new vaccine candidates in preclinical and clinical trials. In contrast, the major structural antibody epitopes remain unclear for the hMPV F protein. Overall, this review will cover recent advances in characterizing the antigenic sites on the RSV and hMPV F proteins.

Respiratory viruses in pediatric necropsies: an immunohistochemical study.

Infections of the respiratory system are responsible for the majority of hospitalizations and deaths in pediatric patients in developing countries. We selected 177 necropsies of pediatric patients who died as a result of serious respiratory infections. The histopathological findings and epidemiological data were reviewed, and lung tissue samples were separated for immunohistochemistry testing. Conventional immunohistochemistry techniques were used to detect viral antigens in formalin-fixed, paraffin-embedded (FF-PE) lung tissue samples using a pool of monoclonal antibodies against respiratory viruses (respiratory syncytial virus, influenza A and B, adenovirus, and parainfluenza 1, 2, and 3 viruses) as primary antibodies. The histopathological findings were classified into bronchopneumonia (BCP) and interstitial pneumonitis (IP) patterns. The immunohistochemistry results were compared with histopathological patterns and epidemiological data. Positive results for viruses were found in 34% and 62.5% of the BCP and IP cases, respectively. Males and infants below 1 year of age were more frequent in the group that had positive results for viruses. Acute enteritis was the main cause of hospitalization and sepsis the most frequent cause of death in this group. A clear seasonal distribution was observed, with the majority of cases occurring in the 2nd and 3rd trimesters (autumn and winter) of each year in the period studied. Immunohistochemistry is an affordable and easy-to-perform method for viral-antigen detection in FF-PE tissue samples. Although BCP is a classic histopathological pattern found in bacterial infections, it is possible that children with serious respiratory infections had concomitant viral and bacterial infections, regardless of their previous immunologic state.

Comparison of the Seeplex reverse transcription PCR assay with the R-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses.

This study evaluated the clinical usefulness of a newly introduced multiplex reverse transcription PCR assay (Seeplex RV; Seegene, Inc., Seoul, Korea) in patients with respiratory symptoms. Fifty clinical respiratory specimens (45 from children, 5 from adults) were tested for 8 viruses (influenza virus type A and B, parainfluenza virus type 1, 2, 3, respiratory syncytial virus type A and B, and adenovirus) by Seeplex RV (S-RV) and R-mix viral culture with immunofluorescence (VC-IF). Forty (80%) of the 50 samples showed concordant results between S-RV and VC-IF; 24 of these showed the same positive and 16 showed the same negative results. Among the 10 discrepant samples, 9 were S-RV-positive and VC-IF-negative. Six were obtained in patients with lower respiratory tract infection. Only 1 sample was VC-IF-positive and S-RV-negative. This patient had pneumonia. In 3 cases, more than 1 virus was identified by S-RV. The total running time of S-RV was 6 hr, which shortens the detection time for the viral presence by 2 workdays compared to VC-IF. In conclusion, S-RV is reliable, rapid, relatively easy to perform, and able to detect more than 1 virus simultaneously. Therefore, implementation of the S-RV assay in clinical laboratories will aid rapid diagnosis and treatment of major viral infections of the respiratory tract.

Mucosal inoculation with an attenuated mouse pneumovirus strain protects against virulent challenge in wild type and interferon-gamma receptor deficient mice.

Protective mechanisms underlying the responses to mucosal vaccination are not yet clearly defined. Using the natural mouse pneumovirus pathogen, pneumonia virus of mice (PVM), we explore responses of wild type and interferon-gamma (IFNgamma) receptor gene-deleted mice to virulent challenge after mucosal vaccination with an attenuated virus strain. Serum neutralizing antibodies develop after intranasal inoculation with 30 pfu of attenuated, replication-competent PVM strain 15, which correlate with diminished gross and microscopic pulmonary pathology and protection from weight loss in response to subsequent challenge with the virulent parent PVM strain J3666. Virus replication in response to challenge was blunted in PVM strain 15 vaccinated mice, as was local production of secretory mediators IFNgamma, TNF-alpha, MIP-1 alpha, and MIP-2. Interestingly, responses of vaccinated IFNgamma receptor gene-deleted mice were indistinguishable from those of the wild type, suggesting that IFNgamma signaling may not be crucial for the generation of adaptive responses to pneumovirus infection in vivo.

Stimulation of pneumovirus-specific CD8+ T-cells using a non-toxic recombinant ricin delivery system.

Internalisation of the plant toxin ricin occurs by retrograde transport which delivers the toxin to the ER where it intersects with the MHC class I system for peptide antigen display. Here, we describe the generation of an inactivated, non-toxic, ricin molecule fused to a peptide which elicits a CD8+ T-cell response in mice directed against pneumonia virus of mice, a pneumovirus related to human respiratory syncytial virus. The ricin fusion elicited a significant T-cell response when delivered by intraperitoneal inoculation in the absence of adjuvent. Challenge experiments showed that the T-cell response resulting from inoculation with the ricin-peptide fusion molecule delayed the onset of virus-induced disease.

Activation and inactivation of antiviral CD8 T cell responses during murine pneumovirus infection.

Pneumonia virus of mice (PVM) is a natural pathogen of mice and has been proposed as a tractable model for the replication of a pneumovirus in its natural host, which mimics human infection with human respiratory syncytial virus (RSV). PVM infection in mice is highly productive in terms of virus production compared with the situation seen with RSV in mice. Because RSV suppresses CD8 T cell effector function in the lungs of infected mice, we have investigated the nature of PVM-induced CD8 T cell responses to study pneumovirus-induced T cell responses in a natural virus-host setting. PVM infection was associated with a massive influx of activated CD8 T cells into the lungs. After identification of three PVM-specific CD8 T cell epitopes, pulmonary CD8 T cell responses were enumerated. The combined frequency of cytokine-secreting CD8 T cells specific for the three epitopes was much smaller than the total number of activated CD8 T cells. Furthermore, quantitation of the CD8 T cell response against one of these epitopes (residues 261-270 from the phosphoprotein) by MHC class I pentamer staining and by in vitro stimulation followed by intracellular IFN-gamma and TNF-alpha staining indicated that the majority of pulmonary CD8 specific for the P261 epitope were deficient in cytokine production. This deficient phenotype was retained up to 96 days postinfection, similar to the situation in the lungs of human RSV-infected mice. The data suggest that PVM suppresses T cell effector functions in the lungs.

Serological monitoring on layer farms with specific pathogen-free chickens.

To monitor the existence of avian pathogens in laying chicken flocks, specific pathogen-free (SPF) chickens were introduced into two layer farms and reared with laying hens for 12 months. SPF chickens were bled several times after their introduction and examined for their sero-conversion to avian pathogens. As a result, antibodies to eight or ten kinds of pathogens were detected in SPF chickens on each farm. Antibodies to infectious bronchitis virus (IBV), avian nephritis virus, Mycoplasma gallisepticum and M. synoviae were detected early within the first month. Antibody titer to IBV suggested that the laying chickens were infected with IBV repeatedly during the experiment on both farms. However, antibodies to infectious bursal disease virus and 6 pathogens were not detected.

Detection and characterization of proteins encoded by the second ORF of the M2 gene of pneumoviruses.

The nucleotide sequence of the M2 gene of pneumonia virus of mice (PVM) was determined. The sequence showed that the gene encoded a protein of 176 amino acids with a predicted molecular mass of 20165 Da from a major ORF, which is smaller than the equivalent proteins encoded by human, bovine and ovine respiratory syncytial (RS) viruses. The PVM M2 protein is conserved, having 41% similarity to the equivalent human RS virus protein. In common with the M2 genes of the RS viruses and avian pneumovirus (APV), the PVM mRNA also contained a second ORF (ORF2) that partially overlaps the first ORF and which is capable of encoding a 98 residue polypeptide. No significant sequence identity could be detected between the putative M2 ORF2 proteins of PVM, APV and the RS viruses. The expression of the M2 ORF2 proteins of the pneumoviruses was investigated by using monospecific antisera raised against GST fusion proteins. Western blot analysis demonstrated the presence of polypeptides encoded by M2 ORF2 of PVM and RS virus corresponding with those predicted by in vitro translation studies, but this was not the case for APV. The PVM polypeptide was present as three distinct products in vivo. The PVM and RS virus polypeptides were also detected in cells by immunofluorescence, which showed that both were present in the cytoplasm with a degree of localization in inclusion bodies. No APV M2 ORF2 protein could be detected in vivo. The RS virus M2 ORF2 polypeptide was shown to accumulate during infection and the potential implications of this are discussed.

Intracellular IFN-gamma expression in natural killer cells precedes lung CD8+ T cell recruitment during respiratory syncytial virus infection.

Natural killer (NK) cells are recruited locally during the initial phases of virus infection and produce cytokines which may affect the subsequent emergence of specific T cells. In this study, cellular responses to primary respiratory syncytial virus (RSV) infection and after vaccination with individual viral proteins were investigated in BALB/c mice using the new NK cell antibody, DX5. Purified DX5+ cells caused lysis of YAC-1 cell targets. DX5+ cells did not express CD8, CD45R or MHC class II antigens. A small proportion of DX5+ cells co-expressed CD4 (10.3%) and CD3 (10.6%). Of the DX5+/CD4+ cells, the majority expressed the alpha/beta T cell receptor and less than 1% expressed the gamma/delta T cell receptor. During infection with RSV, lung DX5+/CD3- NK cells peaked on day 4 of primary infection and were the most numerous subset producing IFN-gamma, as determined by intracellular staining, at this time-point. Less than 1% of the DX5+ cells secreting IFN-gamma were CD4+. In the lungs of mice vaccinated with recombinant vaccinia virus expressing individual RSV proteins, increased NK cell cytotoxicity and IFN-gamma production correlated with increased numbers of CD8+ T cells. Mice with few NK cells subsequently had low CD8+ T cells and developed lung eosinophilia. IFN-gamma-producing NK cells therefore form a substantial component of the early cellular response to virus infection with important potential influences on the subsequent development of specific immunity.

Inhibition of cell fusion by neutralizing monoclonal antibodies to F1 protein of turkey rhinotracheitis virus.

Two monoclonal antibodies (mAbs) with neutralizing activities directed to the fusion (F) 1 glycoprotein of the 8597/CV94 strain of turkey rhinotracheitis (TRT) virus inhibited cell fusion by TRT virus in vitro. In contrast, F1-specific mAbs with no neutralizing activity did not inhibit the cell fusion participating in syncytium formation. These findings indicate that the neutralization epitope is the same as or overlaps the fusion active site.

Protection against turkey rhinotracheitis pneumovirus (TRTV) induced by a fowlpox virus recombinant expressing the TRTV fusion glycoprotein (F).

A recombinant fowlpox virus was produced which expressed the fusion protein (F) of turkey rhinotracheitis virus (TRTV), a pneumovirus. Turkey poults were vaccinated twice, at an interval of 2 weeks, intramuscularly and by wing web on each occasion, with the recombinant or a control fowlpox virus. Two weeks after the second vaccination the poults were challenged superconjunctivally and intranasally with virulent TRTV. A partially protective immune response was achieved; turkeys vaccinated with the F recombinant showed milder clinical signs and 1000-fold less challenge virus was recovered from the nose and trachea compared with turkeys that had been vaccinated with control fowlpox virus. Expression of the F protein induced antibodies which were detectable both by an ELISA and a virus neutralization test. These results show that the immune responses to the F protein play a major role in protection against TRTV and indicate that recombinant viruses expressing the TRTV F protein have potential as vaccines against TRT.