A safe and efficient BCG vectored vaccine to prevent the disease caused by the human Respiratory Syncytial Virus

Abstract

The human Respiratory Syncytial Virus (hRSV) causes lower respiratory tract infections including pneumonia and bronchiolitis. Such infections also cause a large number of hospitalizations and affects mainly newborns, young children and the elderly worldwide. Symptoms associated with hRSV infection are due to an exacerbated immune response characterized by low levels of IFN-γ, recruitment of neutrophils and eosinophils to the site of infection and lung damage. Although hRSV is a major health problem, no vaccines are currently available. Different immunization approaches have been developed to achieve a vaccine that activates the immune system, without triggering an unbalanced inflammation. These approaches include live attenuated vaccine, DNA or proteins technologies, and the use of vectors to express proteins of the virus. In this review, we discuss the host immune response to hRSV and the immunological mechanisms underlying an effective and safe BCG vectored vaccine against hRSV.

Introduction

The human Respiratory Syncytial Virus (hRSV) causes lower respiratory tract infections and a large number of hospitalizations of newborns, young children and elderly worldwide.¹ hRSV can be responsible from mild symptoms, such as cough and rhinitis, to severe symptoms, such as alveolitis, bronchiolitis and pneumonia. Interestingly, although such a disease mainly affects the respiratory system, extrapulmonary manifestations such as encephalitis have been reported.² Recruitment of immune cells to the lungs, impaired production of IFN-γ and lung damage characterize the hRSV infection. Importantly, hRSV-associated pathology is mainly caused by an exacerbation of the host immune response. Further, after hRSV infection an inefficient adaptive response is established, resulting in frequent reinfection of the host throughout life.^3,4 Different virus immune modulatory strategies, such as inhibition of the Type I IFN signaling cascade and interference with T cell activation, among others, have been shown to contribute to this inefficient host response. Up to date, no licensed vaccine to prevent the disease caused by hRSV infection is available for the population. Although several formulations are currently on preclinical development, only a minor fraction of them are under clinical evaluation.⁵ Only an expensive and multi-dose treatment, consisting on injections of a humanized monoclonal antibody, Palivizumab, is available to treat severe cases of hRSV-associated bronchitis or pneumonia. In this review, we discuss the host immune response during hRSV infection and a proposed new vaccine approach that protects against it.

hRSV evasion from host immune system

hRSV infects primary airway epithelial cells and causes an inflammatory cells recruitment to the site of infection. Upon hRSV infection, distal bronchial airway inflammation occurs, thereby prompting to lung damage. Such an inflammatory response fails to achieve a memory response since reinfections in young children and adults are frequent during winter period.⁶ Further, an impairment of the innate response machinery has been reported during hRSV infection, since Interferon (IFN) α/β signaling has been shown to be blocked by both viral non-structural proteins NS1 and NS2 of the hRSV.^7,8 Upon hRSV recognition, the NLRP3 inflammosome is activated through the viral viporin SH, triggering the production of Interleukine (IL) 1β and IL-18.^9,10 Also, it is thought that NS1 and NS2 proteins impair dendritic cell (DC) maturation, which in turn inhibits T cell activation.¹¹ Further, it has been found that hRSV impairs the capacity of DCs to induce T cell activation, making them defective for IL-2 secretion and also up-regulates the expression of surface activation markers.^12,13 Interestingly, a decrease in the polarization of Golgi apparatus in T cells toward hRSV infected-DCs takes place, herein impairing the immunological synapse (IS). The Nucleoprotein (N) of the hRSV seems to be the hRSV protein responsible of such an inhibition of the IS, which in turns causes an impaired T cell activation.^14,15 The synapse is crucial to trigger an effective and specific adaptive response against hRSV. Impairment of T cell activation by hRSV has been described by various researchers is consistent with the reduced production of IFN-γ found in patients with hRSV-induced bronchiolitis and in infected animals.^16-18 Moreover, the low production of IFN-γ causes a weak T helper (T_H) 1 response, which is key for viral clearance. As a result, hRSV infection induces a biased T_H-2 like response characterized by the production of pro-inflammatory cytokines, such as IL-4 and IL-13 (Fig. 1). Further, a direct correlation between the production of IL-13, IL-12p40 and recurrent wheezing in infants with hRSV bronchiolitis has been reported.¹⁹ Such a cytokine production is accompanied by the infiltration of abundant immune cells to the airways, generating inflammation and lung damage. Similarly, upregulation of IL-17 in the lungs was reported upon hRSV infection in mice and it is thought that this cytokine induces mucus production²⁰ and downmodulation of the CD8⁺ T cell response.²¹ Further, in vitro studies suggested that IL-17 could be suppressing T-bet expression impairing IFN-γ secretion.²⁰ IL-17 upregulation, along with an increase of IL-6 and IL-23p19, can lead to a T_H-17 cell differentiation profile.²⁰ Furthermore, Foxp3⁺ T_reg cells have been found to be recruited to the lungs upon hRSV infection.²² The importance of this subset of T cells in the response to hRSV infection has been supported by experiments consisting of CD4⁺Foxp3⁺CD25⁺depleted-mice, which displayed an exacerbation of inflammation after an hRSV challenge.^23,24 Further, the balance between both T_reg and T_H-17 cells has been reported to be associated with the severity of the hRSV infection.²⁵ Along these lines, it has been reported that IL-27 plays an important role at defining the T_H phenotype by suppressing the IL-17-induced polarization.²⁶ Importantly, one of the potentially detrimental host responses during hRSV infection is the induction of IgG1 and IgG3 antibody isotypes, which fail to efficiently neutralize the virus. The hRSV reinfection that occurs throughout the life further underscores the detrimental response of the host to a second exposure. Along these lines, although memory CD8⁺ T cells can be detected upon hRSV challenge, this response seems insufficient to protect against subsequent reinfections.

Figure 1.

Immune response induced by hRSV in absence or presence of the recombinant rBCG-N-hRSV vaccine. hRSV induce an aberrant immune response due to the erratic T cell polarization, producing a high pro-inflammatory and allergic-like response (A). On the other hand, the rBCG-N-hRSV induces an effective immune response that could control the infection through the polarization of CD4⁺ and CD8⁺ T cells and neutralizing antibodies decreasing the recruitment of immune cells such as neutrophils, eosinophils and the mucus secretion (B).

Development of a vaccine against hRSV

In the 1960s, one of the first vaccines candidate for hRSV was the formalin-inactivated vaccine (FI-hRSV). Unfortunately, when the vaccine recipients were exposed to hRSV, they showed increased rates of severe disease, and up to 80% of vaccinated children required hospitalization.^27,28 This immune-mediated enhanced disease was characterized by an excessive eosinophil infiltration into the airways of vaccinated children. This finding of an enhanced disease in the hRSV seronegative infants warned about a vaccine that promotes an enhancement of the pulmonary disease. Therefore, an efficient vaccine against this pathogen should avoid exacerbation of the hRSV-associated inflammation and induce an efficient T_H-1 response characterized by 1) IFN-γ production, 2) hRSV-specific memory CD4⁺ and CD8⁺ T cell response, 3) efficient and neutralizing specific antibodies, preferably of the IgG2a isotype. After FI-hRSV failure, several vaccine strategies have been developed, such as monoclonal antibodies against proteins of hRSV,^29-32 live attenuated hRSV vaccines,^33,34 virus like particles (VLPs),^35,36 and recombinant vaccines using as a vector parainfluenza virus,³⁷ adenovirus^38,39 or Mycobacterium bovis Calmette-Guerin (BCG).^40,41

Mycobacterium bovis BCG as a safe and efficient vector vaccine for hRSV that could be applied to newborns.

Mycobacterium bovis Calmette-Guerin (BCG) is a live, attenuated vaccine that protects against Tuberculosis (TB) and has been used since 1920. Currently, although several TB vaccines approaches are undergoing, BCG is the only licensed vaccine available to protect against TB and is given to newborns in most countries. Several clinical studies provide support to the notion that BCG is a safe and immunogenic vaccine to prevent TB meningitis and miliary disease.⁴² Importantly, BCG induces a CD4⁺ T_H-1 polarized response in neonates by producing adult-like levels of IL-12p70,^43,44 thereby being an attractive approach for the development of vaccines against several pathogens. The use of BCG as a vaccine vector has been previously reported against human immunodeficiency virus (HIV), rotavirus, Plasmodium yoelii, Bortedella pertussis, measles virus and hepatitis B virus.⁴⁵ Our group has designed and developed a recombinant BCG that expresses the nucleoprotein (N) of hRSV (rBCG-N-hRSV). Importantly, immunization with this rBCG-N-hRSV protects of hRSV associated-lung damage, decreases the infiltration of inflammatory immune cells to the lungs and reduces the virus in the lung tissues when mice were infected with hRSV (Fig. 2).^40,46,47 Further, rBCG-N-hRSV induces an early CD4⁺ and CD8⁺ T cell recruitment to the lungs and IFN-γ and IL-17 secretion in response to specific hRSV antigens.⁴¹ Also, no significant IL-4 production was found after rBCG-N-hRSV immunization, herein supporting a T_H-1 polarization.⁴⁰ The lack of protection found in RAG-mice and in recipient mice with IFN-γ blocked,⁴¹ suggested that the vaccine requires an IFN-γ producer T cell repertoire. Further, CD8⁺IFN-γ⁺ T cells were found in the lungs of rBCG-N-hRSV and hRSV-infected mice,⁴⁷ indicating a memory response induced by the vaccine. Interestingly, the nucleoprotein of hRSV in this vectored vaccine is being delivered from APCs to T cells without impairing the IS, as previously shown in the hRSV-infected DCs. Moreover, it has been found that rBCG-N-hRSV elicits long-lasting immune memory in mice.⁴⁷ Vaccination with rBCG-N-hRSV, along with its subsequent infection, not only induces cellular immunity but also a humoral response against the virus with an antibody secretion against several hRSV proteins, including N, F and G (unpublished results). Thus, although the rBCG-N-hRSV is only expressing the nucleoprotein of the virus, antibodies against other proteins of the virus are produced, a phenomenon known as Linked Recognition. Further, hRSV vaccine triggers an IgG isotype switching from IgG1 to IgG2a, which is key for viral clearance (unpublished results). The importance of this humoral response in the immunity against hRSV is supported by a reduced pathology reported in naïve mice transferred with antibodies of vaccinated mice. Also, the rBCG-N-hRSV antibodies generated displayed neutralizing properties, being able to reduce hRSV plaque forming units in infected cells in vitro.

Figure 2.

Proposed mechanism of the rBCG-N-hRSV vaccine that controls the hRSV-infection. Subsequent to the rBCG-N-hRSV infection and the viral infection, the events goes as follows. (1) Immunological synapsis between the APC (Dendritic cells) and T cells. (2) T_H-1 response polarization and secretion of IFN-γ by CD4⁺ and CD8⁺ T cells. (3) Activation of B cells by the T cells. (4) Antibodies secretion by plasma cells with the proper neutralizing isotype. (5) Cytotoxic activity of the CD8⁺ T cell and depletion of infected-cells. (6) A T_H-17 profile induction suggests a possible way of control for the exacerbated T_H-1 response. (7) Possible IL-27 secretion and proliferation of CD4⁺ T cells and induction of IFN-γ secretion. (8) IL-27 secretion could inhibit the T_H-17 response. (9) Relation between the T_H-17⁺ T_reg that might decrease the hRSV-pathology.

The rBCG-N-hRSV is currently the only hRSV vaccine under development that is intended for use in newborns, which is crucial since most of the hospitalized hRSV-infected children are younger than 6 months old. This vaccine could be an useful tool to prevent TB meningitis, miliary disease and hRSV infection in a single immunization event for this risk population. Along these lines, the rBCG-N-hRSV has also shown to be able to produce mycobacterial antigen-specific response as the wild type BCG counterpart.⁴⁷ Besides the capacity to protect from lung pathology, the rBCG-N-hRSV is able to avoid the long-term behavioral impairment in hRSV-infected mice.⁴⁷ These are promising results of an efficient and safe vaccine against the disease caused by 2 major respiratory pathogens, TB and hRSV.

Immune mechanisms accounting for the protection conferred by the rBCG-N-hRSV vaccine

Taking in account all the previously mentioned results observed in the rBCG-N-hRSV immunized mice, we propose the following model as the mechanism for the protection induced by the vaccine against hRSV. Upon rBCG-N-hRSV immunization, nucleoprotein expressed on the BCG vector is processed by DCs and presented to T cells (Fig. 2). Thereafter, proliferation and expansion of CD4⁺ and CD8⁺IFN-γ⁺ T cells specific for the N protein is triggered. After hRSV challenge, these T cells are activated and produce high levels of IL-12 and low levels of IL-10, inducing the differentiation into a T_H-1 profile. We found high levels of IL-17 in vaccinated and infected mice but no inflammation or hypersensitivity. This result could be explained by the T_H-17 cells repertoire that are counteracted by the production of IL-27, which plays a key role in promoting the IFN-γ secretion by CD4⁺ T_H-1 cells and is characterized by inducing the inhibition of the T_H-17 pathology associated with autoimmunity.⁴⁸ Other possible explanation is that IL-17⁺ T_reg cells are diminishing the hRSV-caused inflammation. The CD8⁺IFN-γ⁺ T cells found in vaccinated mice probably display cytotoxicity activity that depletes hRSV-infected cells through the perforin and granzime system. On the other hand, the CD4⁺ T cells present the N antigen and other proteins to B cells, which promotes their differentiation into plasma cells that secrete antibodies against several proteins of the virus with an IgG2a isotype. We have found high levels of antibodies against the nucleoprotein in vaccinated mice before hRSV challenge (unpublished data). Such result indicates that rBCG-N-hRSV could induce memory B cells, which rapidly proliferate and differentiate into plasma cells and after repetitively hRSV exposure, results in high a frequency of hRSV-specific memory B cells. Further, these antibodies neutralize the virus, resulting in a decrease of the viral replication in the lungs. This IgG2a isotype along with the T_H-1 response reduces the recruitment of inflammatory cells to the lungs. In conclusion, the hRSV-associated immunopathology is prevented by rBCG-N-hRSV immunization in mice.

Disclosure of potential conflicts of interest

The authors declare no conflict of interest.

Funding

This work was supported by the Millennium Institute on Immunology and Immunotherapy from Chile (P09/016-F for AMK), CONICYT/FONDECYT POSTDOCTORADO No. 3160249, CONICYT DOCTORADO N°21151028, FONDECYT grants number: 1150862, 1070352, 1050979, 1040349, 1100926, 1110397, 1131012, 1140010, 1140011, 3140455. Biomedical Research Consortium (BMRC 13CTI-21526 for AMK). FONDEF grant D11I1080.