Mycobacterium bovis BCG Given at Birth Followed by Inactivated Respiratory Syncytial Virus Vaccine Prevents Vaccine-Enhanced Disease by Promoting Trained Macrophages and Resident Memory T Cells.

Respiratory syncytial virus (RSV) infects more than 60% of infants in their first year of life. Since an experimental formalin-inactivated (FI) RSV vaccine tested in the 1960s caused enhanced respiratory disease (ERD), few attempts have been made to vaccinate infants. ERD is characterized by Th2-biased responses, lung inflammation, and poor protective immune memory. Innate immune memory displays an increased nonspecific effector function upon restimulation, a process called trained immunity, or a repressed effector function upon restimulation, a process called tolerance, which participates in host defense and inflammatory disease. Mycobacterium bovis bacillus Calmette-Guérin (BCG) given at birth can induce trained immunity as well as heterologous Th1 responses. We speculate that BCG given at birth followed by FI-RSV may alleviate ERD and enhance protection through promoting trained immunity and balanced Th immune memory. Neonatal mice were given BCG at birth and then vaccinated with FI-RSV+Al(OH)3. BCG/FI-RSV+Al(OH)3 induced trained macrophages, tissue-resident memory T cells (TRM), and specific cytotoxic T lymphocytes (CTL) in lungs and inhibited Th2 and Th17 cell immune memory, all of which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented the innate tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. Therefore, BCG given at birth to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants. IMPORTANCE RSV is the leading cause of severe lower respiratory tract infection of infants. ERD, characterized by Th2-biased responses, inflammation, and poor immune memory, has been an obstacle to the development of safe and effective killed RSV vaccines. Innate immune memory participates in host defense and inflammatory disease. BCG given at birth can induce trained immunity as well as heterologous Th1 responses. Our results showed that BCG/FI-RSV+Al(OH)3 induced trained macrophages, TRM, specific CTL, and balanced Th cell immune memory, which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. BCG at birth as an adjuvant to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants.

CpG in Combination with an Inhibitor of Notch Signaling Suppresses Formalin-Inactivated Respiratory Syncytial Virus-Enhanced Airway Hyperresponsiveness and Inflammation by Inhibiting Th17 Memory Responses and Promoting Tissue-Resident Memory Cells in Lungs.

Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalizations. The formalin-inactivated RSV (FI-RSV) vaccine-enhanced respiratory disease (ERD) has been an obstacle to the development of a safe and effective killed RSV vaccine. Agonists of Toll-like receptor (TLR) have been shown to regulate immune responses induced by FI-RSV. Notch signaling plays critical roles during the differentiation and effector function phases of innate and adaptive immune responses. Cross talk between TLR and Notch signaling pathways results in fine-tuning of TLR-triggered innate inflammatory responses. We evaluated the impact of TLR and Notch signaling on ERD in a murine model by administering CpG, an agonist of TLR9, in combination with L685,458, an inhibitor of Notch signaling during FI-RSV immunization. Activation with CpG or deficiency of MyD88-dependent TLR signaling did not alleviate airway inflammation in FI-RSV-immunized mice. Activation or inhibition of Notch signaling with Dll4, one of the Notch ligands, or L685,458 did not suppress FI-RSV-enhanced airway inflammation either. However, the CpG together with L685,458 markedly inhibited FI-RSV-enhanced airway hyperresponsiveness, weight loss, and lung inflammation. Interestingly, CpG plus L685,458 completely inhibited FI-RSV-associated Th17 and Th17-associated proinflammatory chemokine responses in lungs following RSV challenge but not Th1 or Th2, memory responses. In addition, FI-RSV plus CpG plus L685,458 promoted protective CD8+ lung tissue-resident memory (TRM) cells. These results indicate that activation of TLR signaling combined with inhibition of Notch signaling prevent FI-RSV ERD, and the mechanism appears to involve suppressing proinflammatory Th17 memory responses and promoting protective TRM in lungs.IMPORTANCE RSV is the most important cause of lower respiratory tract infections in infants. The FI-RSV-enhanced respiratory disease (ERD) is a major impediment to the development of a safe and effective killed RSV vaccine. Using adjuvants to regulate innate and adaptive immune responses could be an effective method to prevent ERD. We evaluated the impact of TLR and Notch signaling on ERD by administering CpG, an agonist of TLR9, in combination with L685,458, an inhibitor of Notch signaling, during FI-RSV immunization. The data showed that treatment of TLR or Notch signaling alone did not suppress FI-RSV-enhanced airway inflammation, while CpG plus L685,458 markedly inhibited ERD. The mechanism appears to involve suppressing Th17 memory responses and promoting tissue-resident memory cells. Moreover, these results suggest that regulation of lung immune memory with adjuvant compounds containing more than one immune-stimulatory molecule may be a good strategy to prevent FI-RSV ERD.

Natural Killer and CD8 T Cells Contribute to Protection by Formalin Inactivated Respiratory Syncytial Virus Vaccination under a CD4-Deficient Condition.

Respiratory syncytial virus (RSV) causes severe pulmonary disease in infants, young children, and the elderly. Formalin inactivated RSV (FI-RSV) vaccine trials failed due to vaccine enhanced respiratory disease, but the underlying immune mechanisms remain not fully understood. In this study, we have used wild type C57BL/6 and CD4 knockout (CD4KO) mouse models to better understand the roles of the CD4 T cells and cellular mechanisms responsible for enhanced respiratory disease after FI-RSV vaccination and RSV infection. Less eosinophil infiltration and lower pro-inflammatory cytokine production were observed in FI-RSV vaccinated CD4KO mice after RSV infection compared to FI-RSV vaccinated C57BL/6 mice. NK cells and cytokine-producing CD8 T cells were recruited at high levels in the airways of CD4KO mice, correlating with reduced respiratory disease. Depletion studies provided evidence that virus control was primarily mediated by NK cells whereas CD8 T cells contributed to IFN-γ production and less eosinophilic lung inflammation. This study demonstrated the differential roles of effector CD4 and CD8 T cells as well as NK cells, in networking with other inflammatory infiltrates in RSV disease in immune competent and CD4-deficient condition.

The efficacy of inactivated split respiratory syncytial virus as a vaccine candidate and the effects of novel combination adjuvants.

Clinical trials with alum-adjuvanted formalin-inactivated human respiratory syncytial virus (FI-RSV) vaccine failed in children due to vaccine-enhanced disease upon RSV infection. In this study, we found that inactivated, detergent-split RSV vaccine (Split) displayed higher reactivity against neutralizing antibodies in vitro and less histopathology in primed adult mice after challenge, compared to FI-RSV. The immunogenicity and efficacy of FI-RSV and Split RSV vaccine were further determined in 2 weeks old mice after a single dose in the absence or presence of monophosphoryl lipid A (MPL) + CpG combination adjuvant. Split RSV with MPL + CpG adjuvant was effective in increasing T helper type 1 (Th1) immune responses and IgG2a isotype antibodies, neutralizing activity, and lung viral clearance as well as modulating immune responses to prevent pulmonary histopathology after RSV vaccination and challenge. This study demonstrates the efficacy of Split RSV as an effective vaccine candidate.

Respiratory syncytial virus-like nanoparticle vaccination induces long-term protection without pulmonary disease by modulating cytokines and T-cells partially through alveolar macrophages.

The mechanisms of protection against respiratory syncytial virus (RSV) are poorly understood. Virus-like nanoparticles expressing RSV glycoproteins (eg, a combination of fusion and glycoprotein virus-like nanoparticles [FG VLPs]) have been suggested to be a promising RSV vaccine candidate. To understand the roles of alveolar macrophages (AMs) in inducing long-term protection, mice that were 12 months earlier vaccinated with formalin-inactivated RSV (FI-RSV) or FG VLPs were treated with clodronate liposome prior to RSV infection. FI-RSV immune mice with clodronate liposome treatment showed increases in eosinophils, plasmacytoid dendritic cells, interleukin (IL)-4(+) T-cell infiltration, proinflammatory cytokines, chemokines, and, in particular, mucus production upon RSV infection. In contrast to FI-RSV immune mice with severe pulmonary histopathology, FG VLP immune mice showed no overt sign of histopathology and significantly lower levels of eosinophils, T-cell infiltration, and inflammatory cytokines, but higher levels of interferon-γ, which are correlated with protection against RSV disease. FG VLP immune mice with depletion of AMs showed increases in inflammatory cytokines and chemokines, as well as eosinophils. The results in this study suggest that FG nanoparticle vaccination induces long-term protection against RSV and that AMs play a role in the RSV protection by modulating eosinophilia, mucus production, inflammatory cytokines, and T-cell infiltration.

A recombinant anchorless respiratory syncytial virus (RSV) fusion (F) protein/monophosphoryl lipid A (MPL) vaccine protects against RSV-induced replication and lung pathology.

We previously demonstrated that the severe cytokine storm and pathology associated with RSV infection following intramuscular vaccination of cotton rats with FI-RSV Lot 100 could be completely abolished by formulating the vaccine with the mild TLR4 agonist and adjuvant, monophosphoryl lipid A (MPL). Despite this significant improvement, the vaccine failed to blunt viral replication in the lungs. Since MPL is a weak TLR4 agonist, we hypothesized that its adjuvant activity was mediated by modulating the innate immune response of respiratory tract resident macrophages. Therefore, we developed a new vaccine preparation with purified, baculovirus expressed, partially purified, anchorless RSV F protein formulated with synthetic MPL that was administered to cotton rats intranasally, followed by an intradermal boost. This novel formulation and heterologous "prime/boost" route of administration resulted in decreased viral titers compared to that seen in animals vaccinated with F protein alone. Furthermore, animals vaccinated by this route showed no evidence of enhanced lung pathology upon RSV infection. This indicates that MPL acts as an immune modulator that protects the host from vaccine-enhanced pathology, and reduces RSV replication in the lower respiratory tract when administered by a heterologous prime/boost immunization regimen.

Maternal antibodies by passive immunization with formalin inactivated respiratory syncytial virus confer protection without vaccine-enhanced disease.

Maternal immunization of mice with formalin inactivated respiratory syncytial virus (FI-RSV) resulted in the passive transfer of RSV antibodies but not cellular components to the offspring. The offspring born to FI-RSV immunized mothers showed serum RSV neutralizing activity, effectively controlled lung viral loads without vaccine-enhanced disease, did not induce pulmonary eosinophilia, and cytokine producing cells after live RSV infection. Therefore, this study provides evidence that maternal immunization provides an in vivo model in investigating the roles of antibodies independent of cellular components.