PLoS One. 2015 Jul 14;10(7):e0130755. doi: 10.1371/journal.pone.0130755.

Mammalian Cell-Derived Respiratory Syncytial Virus-Like Particles Protect the Lower as well as the Upper Respiratory Tract.

Walpita P1, Johns LM1, Tandon R1, Moore ML2.
  • 1Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
  • 2Department of Pediatrics, Emory University, Atlanta, Georgia, United States of America; Children’s Healthcare of Atlanta, Atlanta, Georgia, United States of America.

 

Abstract

Globally, Respiratory Syncytial Virus (RSV) is a leading cause of bronchiolitis and pneumonia in children less than one year of age and in USA alone, between 85,000 and 144,000 infants are hospitalized every year. To date, there is no licensed vaccine. We have evaluated vaccine potential of mammalian cell-derived native RSV virus-like particles (RSV VLPs) composed of the two surface glycoproteins G and F, and the matrix protein M. Results of in vitro testing showed that the VLPs were functionally assembled and immunoreactive, and that the recombinantly expressed F protein was cleaved intracellularly similarly to the virus-synthesized F protein to produce the F1 and F2 subunits; the presence of the F1 fragment is critical for vaccine development since all the neutralizing epitopes present in the F protein are embedded in this fragment. Additional in vitro testing in human macrophage cell line THP-1 showed that both virus and the VLPs were sensed by TLR-4 and induced a Th1-biased cytokine response. Cotton rats vaccinated with RSV VLPs adjuvanted with alum and monophosphoryl lipid A induced potent neutralizing antibody response, and conferred protection in the lower as well as the upper respiratory tract based on substantial virus clearance from these sites. To the best of our knowledge, this is the first VLP/virosome vaccine study reporting protection of the lower as well as the upper respiratory tract: Prevention from replication in the nose is an important consideration if the target population is infants < 6 months of age. This is because continued virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers. In conclusion, these results taken together suggest that our VLPs show promise to be a safe and effective vaccine for RSV.

PMID: 26172453

 

Supplement

Expression of one or more viral structural proteins from cDNA results in spontaneous assembly of virus-like particles (VLPs) that resemble the real virus morphologically and immunologically. For enveloped viruses like Respiratory syncytial virus (RSV), surface glycoproteins G, and particularly F protein(s) need to be included along with the M protein that is required for morphogenesis; VLP envelope is derived from the plasma membrane of the host. VLPs are safe because they are devoid of any viral genetic material, and therefore not infectious. Because of these properties, VLPs have been successfully used for a number of applications, to study various aspects of virus lifecycle, but particularly importantly, as vaccines. The safety and effectiveness of VLP-based vaccines is clearly evident from the fact that two of them, human papillomavirus (HPV) and hepatitis B virus (HBV) vaccines, are already approved for human use and several are at various stages of development. Therefore, recently the move has been towards generation of potentially safe and effective particulate vaccines with virus-like properties such as virus-like particles (VLPs), virosomes, and synthetic nanoparticles [1].

RSV infection is of public health concern worldwide since it is a major cause of severe lower respiratory illness in infants and premature babies. Sixty to seventy percent of the children are infected with RSV first year of their life and all are infected by 3 years. The Centers for Disease Control considers RSV to be the “most common cause of bronchiolitis and pneumonia in children under 1 year of age in the United States”. RSV epidemics occur annually in winter months in temperate climates. Elsewhere, it depends on the climate. Worldwide, the estimated RSV disease burden in children under five years is over 30 million lower respiratory tract infections, some 3 million hospitalizations, and 160,000 deaths every year. In the USA alone, between 85,000 and 144,000 infants are hospitalized annually. RSV also causes considerable morbidity in the elderly, and the at-risk adults. At present there is no effective treatment or prevention for RSV disease and the use of passive immunoprophylaxis is limited to high risk infants [1, 2].

RSV vaccine has been in the making for over five decades, but none has been licensed to date. Vaccine development for this virus has been difficult for several reasons such as young age at infections and others. The development of a vaccine for RSV has been additionally problematic. Enhanced disease was caused in the 1960 as a result of alum-precipitated formalin-inactivated RSV (FI-RSV) vaccination, specifically in the youngest cohort between 2 and 7 months old. Vaccine recipient children when subsequently encountered wildtype virus were infected, and caused enhanced disease rather than protection; eighty percent of the children were hospitalized, and 2 died. Since then safety concerns have been paramount, and rightfully so. Subsequent studies in mice showed that FI-RSV vaccine produced 1) poor antibody function that failed to neutralize the virus although high antibody titers were detected by ELISA and 2), high levels of lymphocytes in the blood, and high levels of eosinophilia in the lung tissue caused bronchiolitis, and induced a Th2-biased immune response.

In our study we have produced native VLPs in suspension-adapted HEK 293 cells; the VLPs were composed of the two surface glycoproteins G and F, and the matrix protein M, that is required for morphogenesis. In Figure 1, Panel a, we confirmed by western blotting, that the VLPs were indeed composed of these three proteins and that the recombinantly expressed F protein was cleaved intracellularly similarly to the virus-synthesized F protein to produce the F1 and F2 subunits. The precursor protein F0 is difficult to discern, and the F2 fragment is not seen in Figure because of low kD value. An additional F protein band, a trimer at 150kD is clearly seen in the VLPs, and the virus in Panel c. The presence of F1 fragment is critical for vaccine development since all the neutralizing epitopes present in the F protein are embedded in this fragment. The absence of VLP-containing bands in similarly processed “mock’ particles in Panel b confirm VLP specificity. Additional conformation of VLP bands is provided by the equivalently sized M, F and G proteins in the RSV A2 virus in Panel c.

 

FIG1

 

Next, we wanted to evaluate whether VLPs resemble the virus morphologically and immunologically. In Figure 2, we show morphologic similarity between the virus and the VLPs. Material from VLP-containing band from the sucrose gradient (arrow in Fig. 2a), concentrated and viewed by transmission electron microscopy (TEM) after negative staining. Several negatively-stained VLPs with the fringe of surface glycoproteins are clearly visible, and they resemble those reported for the parental virus (Fig. 2b). Immunoreactivity of the particles was verified by staining with polyclonal (Fig. 2c), and RSV F-specific (Fig. 2d) primary antibodies and gold-labelled secondary antibody. (e) Is a negative control where the primary antibody was omitted and VLPs were stained only with the gold-labelled secondary antibody.

 

FIG2

 

To decide whether or not RSV VLPs would induce Th2-biased cytokine response and prime for immunity associated with enhanced respiratory disease, we did the following experiment: The phorbol-myristate-acetate (PMA)-differentiated human macrophage cell line THP-1 were stably differentiated and well characterized. These cells were grown in 24-well plates, and incubated overnight at 37oC. At that time point, the monolayers were infected with RSV A2 strain to achieve moi of 1, and exposed to RSV VLPs at 25μg/ml. LPS, a TLR-4 ligand, was used as a positive control. THP-1 cells treated with media were used as a reference negative control sample. Each reaction was done in duplicate, and with and without the addition of anti TLR-4 antibody. Twenty hrs post-treatment at 37°C, the supernatants were harvested for evaluation of cytokine secretion (IL-4, IL-5, IL-6, IL-12p70, IFN-y, TNF-α, IL-13 and IL-10, and eotaxin) using a Luminex-based multiplex Procarta Cytokine Assay Kit. All tests were conducted at least in duplicate. The results presented in Figure 3 show definitively, that the Th1 cytokine TNF-α, and IL-6 were both upregulated while the Th2 cytokines IL-4, IL-5 and IL-13, and eotaxin were not; IL-10 was in the lower range. These results indicate a Th1-predominant immune response. This Figure shows also that TLR-4 plays a critical role in VLP and virus-induced cytokine response since blockade of TLR-4 signaling with anti TLR-4 antibody resulted in substantial reduction in IL-6, IL-10, and TNF- α. Together, these findings suggest that RSV VLPs and the virus are both sensed by TLR-4. We only assessed TLR-4 as a sensor for VLPs and the virus. For VLPs, other surface sensors like TLR-2 may have played a synergistic role with TLR-4 although this appears unlikely since blocking TLR-4 reduced TNF-α and IL-10 production to the baseline level, and IL-6 production close to baseline. This result suggests that TLR-4 is an essential host cell sensor for RSV VLPs. For RSV however, TNF-α, and IL-6 production was only partially dependent on TLR-4. While TLR-4 is necessary to clear RSV infection effectively, other surface TLRs and several internal host cell-sensors are known to play a role in the induction of innate immunity by the virus.

 

 FIG3

 

We also tested protective efficacy in the cotton rats (CRs) model of RSV disease. Briefly, we had two routes of vaccination, IM (intramuscular) and subcutaneous (SC) and we vaccinated with VLPs alone, and also RSV VLPs adjuvanted with alum and MPLA. We used two doses of VLPs, 25µg per dose three weeks apart, i.e., on day 0 and 21; booster dose was same as the primary dose. Challenge with live RSV A2 virus was three weeks later on day 42 and euthanasia 4 days later on day 46. Our results (Figure 4) showed that CRs immunized with VLPs were not effective and showed low levels of neutralizing antibody. Even this low level of neutralizing immunity cleared the challenge virus in the lung by a small amount. However, such reduction was not meaningful and would not have provided significant protection at this site. Note that there was no reduction of the challenge virus in the nose, and consequently no protection. Thus, adjuvant would be needed. Our adjuvanted VLPs induced potent neutralizing antibody response, and conferred protection in the lung as well as the nose based on substantial virus clearance from these sites. Prevention from replication is an important consideration in infants < 6 months of age. This is because continued virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers.

 

FIG4

 

Importance of the study: To the best of our knowledge, this is the first VLP/virosome vaccine study reporting protection of the lower as well as the upper respiratory tract: Prevention from replication in the nose is an important consideration if the target population is infants < 6 months of age. This is because continued virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers. In conclusion, these results taken together suggest that our VLPs show promise to be a safe and effective vaccine for RSV.

 

References:

  1. Pramila Walpita. Virus-like particle and synthetic nanoparticle-based vaccines for paramyxoviruses, Research Trends, Current Topics in Virology. 2014, Vol.12, 87-97
  2. Walpita P, Johns LM, Tandon R, Moore ML. Mammalian Cell-Derived Respiratory Syncytial Virus-Like Particles Protect the Lower as well as the Upper Respiratory Tract. PLoS One. 2015 Jul 14;10(7):e0130755.

 

 

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