Protection of Ferrets Against Avian Influenza H7N9 Challenge Following Intranasal Dosing of Inert Bacterial Spores

doi:10.21203/rs.3.rs-1767832/v1

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Protection of Ferrets Against Avian Influenza H7N9 Challenge Following Intranasal Dosing of Inert Bacterial Spores

https://doi.org/10.21203/rs.3.rs-1767832/v1

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Background

Influenza is a respiratory infection that continues to present a major threat to human health with ~ 500,000 deaths/year. Continued circulation of epidemic subtypes in humans and animals potentially increases the risk of future pandemics. Vaccination has failed to halt the evolution of this virus and additional "universal" prophylactic approaches should be considered. Naked, "heat inactivated", or inert, bacterial spores have been shown to protect against influenza in murine models.

Methods

Ferrets were administered intranasal doses of inert bacterial spores (DSM 32444^K). Seven days after the last dose animals were challenged with H7N9. Clinical signs of infection and viral shedding were monitored.

Results

Clinical symptoms of infection were significantly reduced in animals dosed with DSM 32444^K. The temporal kinetics of viral shedding were reduced but not abolished,

Conclusion

Taken together nasal dosing using heat-stable spores could provide a simple, cost-effective approach for influenza prophylaxis in both humans and animals.

Innate immunity

influenza

bacterial spores

prophylaxis

Bacillus subtilis

COVID-19 has refocused attention on potential solutions to not only the current pandemic but also to future threats. One candidate that might be considered is the use of bacterial spores. Spores of Bacillus subtilis have been used extensively as vaccine delivery vehicles, either by engineering spores to express antigens or by adsorption of heterologous antigens to the spore surface [1, 2]. With the adsorption approach, it has been shown that heat inactivated H5N1 influenza virions (NIBRG-14 clade) delivered by loaded spores conferred localized immunity as well as protection in murine models of infection [3]. Cross neutralization of other clades as well as antigen sparing were also demonstrated using this approach [3]. Remarkably, inactivated (inert) spores carrying no antigen (naked spores), administered intranasally, also conferred protection to a mouse adapted strain of H5N2 [3]. The underlying mechanism was shown to be that of innate immunity via TLR (Tolllike receptor)-mediated expression of NF-kb and recruitment of NK cells into the lungs together with maturation of DCs [3]. Spores have a number of attributes that may directly or indirectly be involved in this phenomenon. First, they have been shown to activate TLR receptors 2, 4 and 6 [3–5]. Second, spores co-administered with antigens whether by the same route (mucosal) or different routes (systemic antigen - mucosal spores) both augment localized immunity as well as direct balanced antigen-specific Th1-Th2 immune responses [6].

Seasonal A and B viruses circulate among humans with resulting epidemics of acute respiratory disease estimated at 3–5 million cases and up to 650,000 mortalities/year [7, 8]. Infections resulting from zoonotic influenza A viruses can cause severe illness and contribute to the emergence of pandemic strains. Current and recent outbreaks of avian and swine flu not only afflict farmed animals but also increase the risk of transfer to humans and potentially increasing the risk of a future pandemic [9]. It seems appropriate then to evaluate whether the use of inert spores might protect against influenza in a more robust model of influenza infection. The ferret model is considered superior to mouse and guinea pig models since it allows both viral replication without prior virus adaptation as well as transmission, more importantly clinical signs of infection mirror those found in humans [10, 11].

Novel human influenza A virus (IAV) infections during the past decade have included the H7N9 subtype, first isolated from humans in China in early 2013 [12]. In this paper we have investigated the protective efficacy of inert B. subtilis spores in the ferret model of influenza infection using the H7N9 subtype for challenge. We show that intranasal dosing of inert spores showed a significant reduction in clinical signs of disease but did not substantially impact viral shedding.

Spore inoculum

B. subtilis strain DSM 32444 was used for this study. This strain is recommended as safe for human consumption (USA FDA GRAS-notification GRN 000905). Spores were prepared in batch culture (800 mL) and suspensions in dH₂O inactivated by autoclaving and are referred to henceforth as DSM 32444^K. Aliquots (1 mL) were used for animal studies with each lot containing 5 x 10¹⁰ inactivated spore particles (determined by microscopic counting using a Neubauer counting chamber). All sample analysis was conducted using validated methods and all manufacture was conducted in compliance with current Good Manufacturing Practice (WHO GMP) at HURO Biotech (Vietnam),

Virus

A zoonotic avian influenza virus strain A/Anhui/1/13 [H7N9] (accession no. EPI4399507, EPI439509) was used for the infectious challenge [13].

Ferret study

United Kingdom regulations categorize H7N9 as a Specified Animal Pathogens Order (SAPO) 4 and Advisory Committee on Dangerous Pathogens (ACDP) Hazard Group 3 pathogen because it is a notifiable animal disease agent and presents a zoonotic risk, hence the stages of the in vivo experiment involving H7N9 virus were conducted in licensed containment level 3 facilities at the Animal and Plant Health Agency (APHA), Addlestone, Surrey, UK. Two groups of ferrets (Mustela putorius furo; non-albino) each consisting of 8 animals (4 male, 4 female; ~750-1000g) were acclimatized for 7-days prior to treatment. Immediately after this period (Day 0) animals were confirmed free of current influenza infection with HAI titers ≤ 1/5 and absence of viral RNA in nasal wash samples. Animals were housed in groups (n = 4) by sex in open cages. At Day 0, 7, 14 and 21 animals were intranasally administered 0.5 mL of DSM 32444^K (Group 1) or PBS (Group 2) (0.25 mL/nare). For DSM 32444^K this equated to a total of 2.5 x 10¹⁰ spore particles per dose. At Day 28 animals were transferred to BSL3 facilities and all were challenged intranasally (0.5 mL/nostril) with AIV (A/Anhui/1/12 [H7N9]) at a titer of 1 x 10⁷ TCID₅₀/mL (tissue culture infectious dose). The viral titer was back-titrated following inoculation by TCID₅₀ to ensure that an accurate viral titer had been administrated.

Weight and temperatures were taken daily from all ferrets. Nasal washes were performed every other day-post infection and detection and quantification of viral RNA performed by RT-qPCR. On Day 42 (12 days post-infection, d.p.i.) or at a defined endpoint animals were euthanized and blood taken for serological analysis.

Assessment of clinical scores

All ferrets were assessed daily for clinical scores (Additional File 1). Weights and temperatures were taken at daily intervals and body temperatures were monitored using subdermal microchips implanted in the nape of the ferret.

Sampling and analysis

For nasal washes, ferrets were anaesthetized using isoflurane (IsoFlo, Zoetis) and 1 mL (0.5 mL/nare) of Dulbecco's PBS (Sigma) was administered intranasally and used to lavage the nasal cavity as described previously [13]. The nasal washes were collected into 2 mL Eppendorf tubes and stored at -80ºC until required. Blood was obtained both prior to infection via venipuncture and post-infection via cardiac puncture under terminal anesthesia. Blood was stored at 4ºC to facilitate clotting, centrifuged at 1,000g and serum aliquoted into fresh tubes.

Serology

Serum was treated with receptor destroying enzyme (RDE) (supplied and validated by APHA) and heat-treated at 56ºC for 30 min, seroconversion to AIV H7N9 was assessed using the hemagglutination inhibition assay (HAI) as previously described [13]. Beta-propiolactone (BPL) (97%, Sigma) inactivated homologous A/Anhui/1/13 [H7N9] antigen was used to quantify hemagglutination inhibition titers (HITs). HITs ≤ 1/5 were considered negative.

RT-qPCR analysis

RNA was extracted from ferret nasal wash samples using the KingFisher™ Flex Purification System and MagMax™-96 Total RNA Isolation Kit technology (Invitrogen) and eluted in 90 mL of DEPC-treated RNase free H₂O. Influenza A virus RNA was detected using an influenza A specific RT-qPCR targeting the Matrix-gene as described elsewhere [14]. Virus titer was determined by using a serially diluted standard curve generated from RNA extracted from the homologous virus (A/Anhui/1/13 [H7N9]) at a starting titer of 1 x 10⁷ TCID₅₀/mL, as previously described [13]. The final viral titer in each sample was presented as relative equivalency units (REUs), calculated based on Cq values and extrapolated from the standard curve. The limit of detection was a value of ≥ 36.00 Cq (≥ 7.012 REUs) based on the threshold for influenza A virus diagnostic testing [14].

Symptomatic Protection to H7N9 AIV Infection

Prior to challenge animals administered four intranasal doses of DSM 32444^K or PBS (control group) exhibited no clinical signs including weight loss or elevated temperatures (Additional File 2). After the last dose of DSM 32444^K or PBS animals were infected (intranasal) with H7N9. One ferret (no. 323) in the control group was euthanized at 7-days post-infection (dpi). on welfare grounds due to prolonged weight loss, loss-of-appetite and dehydration. For remaining animals, clinical signs were monitored and graded (Additional File 1). Infection with H7N9 virus typically induces a range of clinical signs including lethargy, loss-of-appetite, change in fur appearance, dehydration and elevated temperatures. Animals in the control group exhibited more severe clinical signs compared to animals receiving DSM 32444^K spores (Fig. 1). All ferrets exhibited mild and transient pyrexia starting from 1 dpi. with a maximum of a 2^oC increase from the predefined baseline (Fig. 2A). Elevated temperatures returned to baseline levels from 4 to 6 dpi for all groups. Pyrexia was most pronounced in the control treated female group and at 2 dpi DSM 32444^K-treated animals exhibited a significantly (p < 0.05) lower body temperature. Most ferrets exhibited weight loss with a 20% reduction in weight from baseline data between 1 and 5 dpi. (Fig. 2B). The control treated groups exhibited significantly (p < 0.0001) greater weight loss compared to DSM 32444^K-treated ferrets (Fig. 2B). Taken together, this study shows that intranasal administration with spores of DSM 32444^K reduced symptoms of infection caused by a zoonotic influenza A virus.

Protection Against Viral Shedding

All ferrets shed detectable viral H7N9 RNA between 2 x 10⁸ and 2 x 10⁴ REU.mL^− 1 (REU, relative equivalency units) from their nasal cavities at 2 dpi. indicating that all animals had become productively infected with H7N9 (Fig. 2C). Shedding was detectable in all ferrets, above the limit of detection, until 10 dpi for the control group and 6 dpi. for the DSM 32444^K-treated group (Fig. 2C). However, there was no statistical difference in viral shedding between groups using one way ANOVA at any time point following infection. In sum, this data suggests that prior treatment with DSM 32444^K does not alter the titer of virus being shed or overall virus load following infection with avian influenza A. Although not statistically significant, treatment with DSM 32444^K did appear to reduce the window of virus shedding.

Although rRT-qPCR is highly sensitive and was used here to detect viral RNA it is unable to provide insight into the titers of infectious virus present within the sample for which egg isolation is traditionally used. Despite this limitation it appears that dosing with spores has little if any impact on viral shedding. This indicates that protection most likely occurs by an unspecific mechanism such as innate immunity.

Effect of DSM 32444^K on seroconversion to Influenza A virus

Serum was collected from all ferrets at 14 dpi. (with the exception of the one euthanized animal). All ferret serum was treated with receptor destroying enzyme and tested via the haemagglutinin inhibition assay using homologous H7N9 antigen. All ferrets exhibited seroconversion to the H7N9 antigen (HAI titers > 29) confirming active infection.

This study strengthens an earlier finding [3] that intranasal dosing of mice with inert B. subtilis spores has the ability to reduce symptoms of influenza. Here we used the ferret model of influenza which is generally considered as more informative since it more closely mimics infection in humans presenting both the symptoms of disease as well as replication and shedding of virus. Our data shows that pre-treatment with inert spores of B. subtilis shows a clear reduction in the virulent and symptomatic stages of influenza infection. However, shedding of the virus was not significantly affected. That protection to symptoms could be achieved in the more robust ferret model is encouraging. Firstly, it agrees with our earlier mouse study (which measures only clinical signs and symptoms rather than viral shedding) and secondly, it shows that inert spores have potential utility for controlling this viral disease in humans and/or in animals. It seems likely that with further refinement and modification of either dose or dosing regimen increased levels of protection might be achieved. As reported previously [3] the underlying mechanism is most probably that of innate immunity.

Innate immunity plays an important role in influenza [15]. One of the key mechanisms that evokes innate immunity is interaction of heterologous ligands with pattern recognition receptors, most typically being Toll-like receptors (TLRs), displayed on the surface of host cells. A number of TLR-agonists have been shown to have efficacy in preventing influenza disease [16–19]. Inert spores then, add to the number of TLR-agonists that are able to prevent symptoms of influenza. In this case though the use of spores has a number of unique attributes that lend themselves to further development. Bacterial spores are produced simply and cost-effectively using bioreactors and spores are commercially produced as probiotics or animal feed products [20]. The majority of Bacillus strains used are considered safe for human consumption [21] and of course humans and animals are exposed to these ubiquitous and environmentally-acquired bacteria on a daily basis with daily exposure estimated at 10⁴-10⁵ spores/day [22]. Studies in mice have shown that intranasal administration of inactivated spores leads to complete clearance of bacteria from the lungs in as little as 24 h [2].

Inert bacterial spores have been shown to have efficacy in reducing the symptoms of influenza in a robust animal model. Influenza is a disease that has global impact both for humans and animals. Considering the specter of future pandemics it seems prudent to consider the use of inert spores for a potential, low cost and pragmatic, prophylactic measure for control of influenza infection in animals and possibly also in humans. That the underlying mechanism is innate immunity then it is of course also of interest to know whether inert spores might have a broader spectrum of efficacy against other viral pathogens.

Ethics approval. Work was conducted at the Animal and Plant Health Agency (APHA) under UK Home Office licence PP9878849 for which ethical approval was obtained from the APHA's AWERB (Animal Welfare and Ethical Review board).

Consent for publication. Not applicable

Availability of data and materials. All data generated or analysed during this study are included in this published article (and its supplementary information files).

Competing interests. SMC is CEO of SporeGen Ltd

Funding. This grant was supported by an Innovate UK grant (IUK: 72725) to SporeGen Ltd. and Destiny Pharma plc.

Authors contributions. SMC, DH, WL and HAH wrote the manuscript. HAH designed the experimental study. PK and DH conducted statistical analysis. CD, LV, WF and Y-LT conducted exp studies.

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Competing interest reported. SMC is CEO of SporeGen Ltd

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Version 1

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You are reading this latest preprint version

Protection of Ferrets Against Avian Influenza H7N9 Challenge Following Intranasal Dosing of Inert Bacterial Spores

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Spore inoculum

Virus

Ferret study

Assessment of clinical scores

Sampling and analysis

Serology

RT-qPCR analysis

Results

Symptomatic Protection to H7N9 AIV Infection

Protection Against Viral Shedding

Effect of DSM 32444^K on seroconversion to Influenza A virus

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Protection of Ferrets Against Avian Influenza H7N9 Challenge Following Intranasal Dosing of Inert Bacterial Spores

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Spore inoculum

Virus

Ferret study

Assessment of clinical scores

Sampling and analysis

Serology

RT-qPCR analysis

Results

Symptomatic Protection to H7N9 AIV Infection

Protection Against Viral Shedding

Effect of DSM 32444K on seroconversion to Influenza A virus

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Effect of DSM 32444^K on seroconversion to Influenza A virus