Intranasal Respiratory Syncytial Virus Challenge Research Articles

A Recombinant MVA-Based RSV Vaccine Induces T-Cell and Antibody Responses That Cooperate in the Protection Against RSV Infection.

Respiratory syncytial virus (RSV) causes a respiratory disease with a potentially fatal outcome especially in infants and elderly individuals. Several vaccines failed in pivotal clinical trials, and to date, no vaccine against RSV has been licensed. We have developed an RSV vaccine based on the recombinant Modified Vaccinia Virus Ankara-BN® (MVA-RSV), containing five RSV-specific antigens that induced antibody and T-cell responses, which is currently tested in clinical trials. Here, the immunological mechanisms of protection were evaluated to determine viral loads in lungs upon vaccination of mice with MVA-RSV followed by intranasal RSV challenge. Depletion of CD4 or CD8 T cells, serum transfer, and the use of genetically engineered mice lacking the ability to generate either RSV-specific antibodies (T11µMT), the IgA isotype (IgA knockout), or CD8 T cells (β2M knockout) revealed that complete protection from RSV challenge is dependent on CD4 and CD8 T cells as well as antibodies, including IgA. Thus, MVA-RSV vaccination optimally protects against RSV infection by employing multiple arms of the adaptive immune system.

Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant.

Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, TH1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens.

Vaccination with human papillomavirus pseudovirus-encapsidated plasmids targeted to skin using microneedles.

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.

Respiratory syncytial virus (RSV) fusion protein expressed by recombinant Sendai virus elicits B-cell and T-cell responses in cotton rats and confers protection against RSV subtypes A and B

The respiratory syncytial virus (RSV) is a serious pediatric pathogen for which there is currently no clinically approved vaccine. This report describes the design and testing of a new RSV vaccine construct (rSV-RSV-F), created by the recombination of an RSV F sequence with the murine parainfluenza virus-type 1 (Sendai virus, SV) genome. SV was selected as the vaccine backbone for this study, because it has previously been shown to elicit high-magnitude, durable immune activities in animal studies and has advanced to human safety trials as a xenogenic vaccine for human parainfluenza virus-type 1 (hPIV-1). Cells infected with the recombinant SV expressed RSV F protein, but F was not incorporated into progeny SV virions. When cotton rats were inoculated with the vaccine, high-titer RSV-binding and neutralizing antibodies as well as interferon-γ-producing T-cells were induced. Most striking was the protection against intra-nasal RSV challenge conferred by the vaccine. The rSV-RSV-F construct was also tested as a mixture with a second SV construct expressing the RSV G protein, but no clear advantage was demonstrated by combining the two vaccines. As a final analysis, the efficacy of the rSV-RSV-F vaccine was tested against an array of RSV isolates. Results showed that neutralizing and protective responses were effective against RSV isolates of both A and B subtypes. Together, experimental results encourage promotion of this recombinant SV construct as a vaccine candidate for the prevention of RSV in humans.

Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis.

A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene-gun immunization of BALB/c mice with this construct induced an antigen-specific CD8+ T-cell memory. After intranasal RSV challenge, accelerated CD8+ T-cell responses were observed in pulmonary lymph nodes and virus clearance from the lungs was enhanced. The construct induced weaker CD8+ T-cell responses than those elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance.

Inhibition of T1/ST2 during respiratory syncytial virus infection prevents T helper cell type 2 (Th2)- but not Th1-driven immunopathology.

T cells secreting interleukin (IL)-4 and IL-5 (T helper cell type 2 [Th2] cells) play a detrimental role in a variety of diseases, but specific methods of regulating their activity remain elusive. T1/ST2 is a surface ligand of the IL-1 receptor family, expressed on Th2- but not on interferon (IFN)-gamma-producing Th1 cells. Prior exposure of BALB/c mice to the attachment (G) or fusion (F) protein of respiratory syncytial virus (RSV) increases illness severity during intranasal RSV challenge, due to Th2-driven lung eosinophilia and exuberant Th1-driven pulmonary infiltration, respectively. We used these polar models of viral illness to study the recruitment of T1/ST2 cells to the lung and to test the effects of anti-T1/ST2 treatment in vivo. T1/ST2 was present on a subset of CD4(+) cells from mice with eosinophilic lung disease. Monoclonal anti-T1/ST2 treatment reduced lung inflammation and the severity of illness in mice with Th2 (but not Th1) immunopathology. These results show that inhibition of T1/ST2 has a specific effect on virally induced Th2 responses and suggests that therapy targeted at this receptor might be of value in treating Th2-driven illness.

Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses.

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.

Challenge of BALB/c mice with respiratory syncytial virus does not enhance the Th2 pathway induced after immunization with a recombinant G fusion protein, BBG2NA, in aluminum hydroxide.

The polypeptide of aa 130-230 of the G protein (G2Na) of respiratory syncytial virus (RSV) was fused to BB, the albumin-binding region of streptococcal G protein, producing BBG2Na, which induced protective immune responses in rodent models. Evaluation of the immune response in mice immunized with BBG2Na in the adjuvant alhydrogel revealed high amounts of interleukin (IL)-5 and some IL-4 in splenocytes restimulated in vitro. This is compatible with a Th2 response. The activation of the Th2 pathway in such mice was further supported by the detection of IL-5 and G2Na-specific IgE in vivo. Of interest, in contrast to immunization with formalin-inactivated RSV, immunization of mice with BBG2Na followed by intranasal RSV challenge did not lead to increased production of IL-5- or G2Na-specific IgE. However, IgG1- and IgG2a-specific antibodies were boosted. These results demonstrate that the Th2 pathway is not enhanced by RSV challenge in BBG2Na-immunized mice.

Efficacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret model.

In the absence of an adequate small animal model for testing the efficacy of adenovirus-vectored respiratory syncytial virus (RSV) vaccines, a ferret model was established for this purpose. Recombinant adenovirus types 4, 5 and 7 expressing the RSV fusion glycoprotein (F), the attachment glycoprotein (G) or both F and G were constructed previously. These recombinants contain a deletion of a large portion of the E3 region of the respective adenovirus vector. In addition, an Ad7(E3+)F recombinant virus which contains an intact E3 region was constructed to assess whether E3 region functions might enhance vaccine immunogenicity. Evaluation of these viruses in the ferret model demonstrated that Ad4 and Ad5 recombinants, administered intranasally to ferrets, induce stronger seroresponses to RSV than do Ad7 recombinant viruses. Ad7(E3+)F did not show enhanced immunogenicity relative to E3-deleted recombinant viruses. However, measurement of RSV infectivity in nasal washes, following intranasal RSV challenge, showed that five different vaccination regimens, Ad7F/Ad4F, Ad7G/Ad4G, Ad7FG/Ad4FG, Ad4F/Ad7(E3+)F and Ad5F/Ad4F, protected ferrets from RSV infection in a dose-dependent manner.

Evaluation in chimpanzees of vaccinia virus recombinants that express the surface glycoproteins of human respiratory syncytial virus

The immunogenicity and protective efficacy of recombinant vaccinia viruses that express the two major protective antigens of human respiratory syncytial virus (RSV), the F and G glycoproteins, were evaluated in chimpanzees. In previous studies in rodents and monkeys the F and G proteins expressed by the same recombinants were highly immunogenic and induced high levels of resistance to RSV replication following subsequent challenge. In contrast, in chimpanzees, a single intradermal immunization induced only moderate levels of F and G-specific serum antibodies as measured by an enzyme-linked immunosorbent assay, and these antibodies did not efficiently neutralize RSV infectivity in vitro. This poor antibody response in chimpanzees to the F and G glycoproteins occurred despite efficient replication of the vaccinia virus vector as evidenced by lesion size and serum antibody response to vaccinia virus. Upon intranasal RSV challenge, it was observed that prior immunization with the F and G recombinants effected only a marginal reduction in the magnitude and duration of RSV shedding from the nose and trachea and did not reduce illness. However, the RSV challenge induced a strong secondary antibody response, resulting in very high titres (> 8000 reciprocal mean titre) of serum neutralizing antibodies. The poor protective efficacy observed here is discussed with regard to the permissiveness of the chimpanzee to RSV replication, the general requirements for effective immunization against RSV, and the limitations of experimental animals for evaluating candidate RSV vaccines.

Immunoglobulin administration and ribavirin therapy: efficacy in respiratory syncytial virus infection of the cotton rat.

We studied the effects of combined administration of human immunoglobulin (IVIG) and ribavirin aerosol on respiratory syncytial virus (RSV) infection in cotton rats (Sigmodon hispidus). Cotton rats assigned to receive combined therapy were administered Gamimune, a preparation of purified IVIG with a high titer of anti-RSV neutralizing activity, intraperitoneally 24 h prior to intranasal RSV challenge and then treated with ribavirin aerosol 3 days after challenge. Lung viral titers from these cotton rats (geometric mean titers [GMT] log10 = 0.15 +/- 0.5) were lower than titers from untreated animals (GMT, log10 = 3.7 +/- 0.6) and animals treated with either IVIG alone (GMT, log10 = 1.8 +/- 0.9) or ribavirin alone (GMT, log10 = 1.9 +/- 1.1). Only one of 12 cotton rats treated with both IVIG and ribavirin had a demonstrable titer of virus after RSV challenge. When IVIG administration was delayed until day 3 after virus challenge, lung viral titers were still lowest in animals receiving both IVIG and ribavirin. In comparison, there was no additive antiviral effect between IVIG and ribavirin against RSV infections of HEp-2 cells in vitro. Pathologic changes on histologic examination of pulmonary tissues from animals challenged with RSV were least prominent in animals treated with both IVIG and ribavirin. Despite the apparent absence of in vitro additive antiviral effect, combined use of IVIG and ribavirin was more efficacious against RSV infection in the cotton rat than use of either agent alone.