Paradoxical modulation of influenza by intranasal administration of non-replicating adenovirus particles

Respiratory mucosal infection by airborne microbes is a common event that occurs every day. We report here that intranasal administration of non-replicating adenovirus (Ad) particles to mice could either confer rapid protection against influenza virus (IFV) challenge independent of adaptive immunity, or exacerbate influenza by triggering rapid death. The life-or-death outcome hinges on the time interval between Ad administration and IFV challenge in conjunction with specific mouse/IFV strains. Intranasal instillation of Ad particles 1–47 days prior to IFV challenge conferred rapid protection against influenza in Balb/c mice whereas exposure to Ad 39 days prior to challenge with a specific IFV strain or 1 day post-challenge with that IFV strain induced rapid death in C57BL/6 mice. Notably, consecutive administrations of Ad prior to IFV challenge conferred a synergy in triggering a potent anti-influenza state; even a detrimental Ad exposure 39 days before challenge with the deadly IFV strain was reversed to a beneficial one by subsequent Ad boosts. Results revealed an intricate relationship between infection and innate immunity that is a linchpin around which effects revolve from protective immunity to collateral damage. It is urgent to repeat the experiments with an expanded scope for characterizing the status that defines susceptibility or resistance to IFV infection and subsequently reveal the underlying mechanisms. Whether broad heterologous protective effects induced by AdE and adaptive immunity elicited by vaccination could confer synergy during mitigation of a pandemic remains to be seen.

This is a Registered Report and may have an associated publication; please check the article page on the journal site for any related articles. E1/E3-defective human adenovirus serotype 5 without exogenous genes; "E" stands for "empty") particles 1 day prior to A/California/04/2009 (CA04; mouse-adapted pandemic 2009 H1N1 swine IFV kindly provided by Dr. Robert G. Webster) challenge conferred seemingly mild protection of C57BL/6 mice against influenza whereas exposure to AdE 39 days prior to CA04 challenge or 1 day post-CA04 challenge induced rapid IFV-mediated death, more rapidly than their counterparts that were challenged with CA04 only (Fig 1A). Notably, consecutive administrations of AdE in conjunction with AdCA04HA1 (Ad vector encoding a codonoptimized CA04 HA1 gene [synthesized at GenScript]) prior to IFV challenge conferred a synergy in triggering a potent anti-influenza state ( Fig 1A); even a lethal AdE exposure 39 days prior to CA04 challenge was reversed to a beneficial one by subsequent AdE/AdCA04HA1 boosts, as shown by significant mitigation of IFV-induced body-weight loss (Fig 1B). Surprisingly, AdE induced an anti-influenza state against live A/Puerto Rico/8/34 (PR8; H1N1; VR-95, ATCC) IFV in Balb/c mice in a similar but different manner [1]. Results suggested that nonreplicating AdE may be endowed with the potential for development into a broad-spectrum antiviral drug that could confer weeks-long heterologous protection of hosts against unrelated mucosal viruses in a wide variety of disease settings without impairment by drug resistance over time, because AdE most likely induces a broad-spectrum antiviral state by remodeling the airway into an Ad territory that impedes other viruses' activities within the respiratory tract without attacking unrelated viruses directly. Since administration of AdE post-IFV challenge either conferred no protection of Balb/c mice [1] or exacerbated influenza in C57BL/6 mice (Fig 1), it is not yet feasible to mobilize AdE-mediated innate immunity toward beneficial post-exposure therapy of influenza. If our hypothesis should be correct, AdE could be developed into a post-exposure antiviral drug by mitigating the initial pro-IFV arm of innate immunity induced by IFV infection. First and foremost, we have to repeat these experiments in greater detail for determining the specific parameters that tip the scale toward proinfluenza and anti-influenza states, respectively.

Hypothesis
It is conceivable that airborne mucosal viruses may constantly compete for territories within human and animal airways. When Ad infects the airway, it may initially program innate immunity into a pro-Ad response that impedes subsequent activities of other viruses (e.g., IFV) in the airway as suggested by our findings [1], until the pro-Ad response is later disabled by anti-Ad immunity. Likewise, a pro-IFV arm of innate immunity could be activated immediately post-IFV infection that incapacitates Ad if Ad should be introduced into the airway post-IFV infection. It would be of medical importance to characterize different arms of innate immunity activated by infection with different viruses in the airway, not only for understanding virus-host and virus-virus interactions but also for paving the way for the development of a new generation of broad-spectrum antiviral drugs that will not be impaired by drug resistance over time in a wide variety of disease settings. The urgency to develop broad-spectrum strategies against pandemics caused by unknown viruses in the future has been brought to a clear focus by the ongoing SARS-CoV-2 pandemic that is ravaging nearly the entire world with no end in sight. Although broad heterologous protective effects have been induced by a number of live vaccines, it is conceivable that they may be elicited via different mechanisms as some of the live vaccines confer beneficial protection only in neonates whereas others may protect adults [2]. Whether broad heterologous protective effects induced by different agents could be coordinated to confer synergy as a powerful tool in the public health arsenal against a pandemic remains to be determined. studies will be supported by UNTHSC (Fort Worth, Texas, USA) and a grant from Flu Lab and Center for Open Science (FL-COS). The funders have provided support in the form of salaries (KBP and IVI), laboratory space (IVI and UNTHSC), equipment and supplies (IVI, UNTHSC, FL-COS), as well as core and animal facilities (IVI and UNTHSC), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:
The author is the CEO and Founder of VaxDome Inc. (VaxDome) and has declared that no competing interests exist. The author's commercial affiliation with VaxDome does not alter his adherence to all PLOS ONE policies on sharing data and materials. The following issued patents are associated with the early phase of this research: U.S. patent (Patent No. 10,183,069); Mexico patent (Patent No. MX/a/2013/010794); Europe patent (Patent No. EP2688588); Japan patent (Patent No. 6,177,981).

Fig 1. Timing of intranasal administration of nonreplicating Ad particles determines whether an influenza virus challenge is ameliorated or exacerbated in mice.
Purified AdE and AdCA04HA1 particles as well as CA04 IFV particles were inoculated into young (2-3 months old) female C57BL/6 mice by intranasal instillation at different time points as described [1]. Mice were monitored daily for survival with body weights recorded every other day postchallenge. (A) Survival rates post-CA04 challenge; % survival was determined by taking the number of mice on Day 0 as 100%. (B) Body weight loss post-CA04 challenge; post-challenge body weights are presented as mean % body weight by taking the body weight of individual mice on Day 0 as 100%. Buffer-1 (negative control group inoculated with virus-free A195 buffer only), A195 buffer was instilled into the nostril one day prior to CA04 challenge; AdE-39, AdE particles were instilled into the nostril 39 days prior to CA04 challenge; AdE-1, AdE particles were instilled into the nostril 1 day prior to CA04 challenge; AdHA-1, AdCA04HA1 particles were instilled into the nostril 1 day prior to CA04 challenge; Ad39-2-1, AdE particles were instilled into the nostril 39 and 2 days prior to CA04 challenge, followed by instillation of AdCA04HA1 particles 1 day prior to CA04 challenge; AdE+1, AdE particles were instilled into the nostril 1 day after CA04 challenge; parentheses represent the number of animals in each group. AdE and AdCA04HA1 particles were instilled into the nostril at a dose of 1.6X10 8 infectious units (ifu) in a volume of 0.05 ml per mouse; CA04 particles were instilled into the nostril at a dose of 8X10 3 plaque-forming units (pfu) in a volume of 0.05 ml per mouse, which was equivalent to approximately 1xLD 50 in this experiment. Statistical analysis was performed using GraphPad Prism version 8.4.3 (686) (GraphPad Software, San Diego, CA). Log-rank tests were performed for comparing Kaplan-Meier survival curves; the protection afforded in AdE-39 group (P = 0.0002), AdE+1 group (P = 0.0055), and Ad-39-2-2 group (P = 0.0131) reached statistical significance when compared to that of the Buffer-1 control group. Mice in the Ad-39-2-1 group significantly gained body weight over time when compared to their counterparts in the Buffer-1 control group (P = 0.0492; unpaired t-test). Statistical significance was set at P<0.05.

Rationale
Intranasal administration of AdE particles into Balb/c mice 1, 2, 22, or 47 days prior to IFV challenge induced an anti-influenza state that conferred protection against a lethal challenge by live PR8 or CA04 IFV, whereas inoculation of AdE post-IFV challenge conferred neither mitigation nor exacerbation of influenza [1]. Intranasal administration of AdE particles into C57BL/6 mice induced a similar but different anti-influenza state (Fig 1). While AdE inoculation 1 day prior to CA04 challenge induced a seemingly mild (statistically insignificant) antiinfluenza state in C57BL/6 mice, AdE administration either 39 days prior to CA04 challenge (P = 0.0002) or 1 day post-CA04 challenge (P = 0.0055) exacerbated influenza by killing C57BL/6 mice more rapidly than their counterparts challenged with CA04 only; and the lethal exposure to AdE 39 days prior to CA04 challenge could be reversed to a beneficial one by subsequent Ad boosts prior to CA04 challenge as shown by 100% survival rate (P = 0.0131) ( Fig  1A) in conjunction with remarkable body-weight gain (P = 0.0492) in the face of IFV infection ( Fig 1B). Similar to AdE, administration of AdCA04HA1 one day prior to CA04 challenge did not change survival rates significantly. It is unclear whether the AdE-mediated exacerbation of influenza at specific time points (Fig 1) was due to the C57BL/6 mouse strain, the CA04 IFV strain, or both. Results implied that random infection of airway by airborne microbes at varying time points may unpredictably ameliorate or exacerbate influenza or diseases caused by other airborne viruses (e.g., coronavirus; respiratory syncytial virus; etc.), and consecutive nasal spray of a benign, nonreplicating, bioengineered virus-derived particle such as AdE could rapidly confer protection of the host against IFV and possibly other unrelated pathogens as well by remodeling the airway into an Ad territory that impedes other viruses' activities. Since AdE-induced anti-influenza state in Balb/c mice slowly declined away after 6 weeks post-administration [1], this novel modality may emerge as a complement to vaccination by conferring broad protection against unrelated pathogens before vaccination-elicited adaptive immunity against a specific pathogen is protective enough [3]. Notably, the safety profile and potency of an Ad-vectored nasal influenza vaccine has been demonstrated in human subjects during a human Phase I clinical trial [4]; it is thus conceivable that there should be no major safety concern for the AdE-mediated therapy option to enter human clinical trials after animal studies pan out. In these studies, we will specifically determine the parameters that tip the scale toward pro-influenza and anti-influenza states, respectively.

Specific aim #1: Determination of the parameters that tip the scale toward an anti-influenza state or exacerbation of influenza
In these studies, we will determine whether exacerbation of influenza by AdE particles is due to the mouse strain, the CA04 IFV strain, or both. AdE particles will be produced, purified, and instilled intranasally into Balb/c and C57BL/6 mice, respectively, at different time points. As shown in Table 1, mice will be challenged by intranasal administration of live PR8 or CA04 IFV as described [1].
If our preliminary data should be reproducible and confirmed during these Stage I studies, we will establish a unique animal model that could enable the characterization of specific innate immunity signatures for predicting susceptibility or resistance to IFV infection. To test our driving hypothesis that AdE activates specific arms of innate immunity that impede IFV growth in the respiratory tract [5], we will analyze the lung during Stage II studies in search of biomarkers that may represent susceptibility or resistance to IFV infection. Lung samples will be collected at different time points from control, AdE-, IFV-, and AdE/IFV-infected mice; labeled antibodies against specific biomarkers ( Table 2) will be used to analyze the panel of immune cells within the lung at varying time points. We will perform flowcytometric analysis of single cell suspensions of the lung to reveal local immunological changes at the portal whereby airborne viruses invade; activation of innate immune cell populations will be measured using CD69 as a marker of activation as described [6]; the % of activated natural killer (NK) cells (NK1.1 + /CD3 -), NKT cells (NK1.1 + /CD3 + ), B cells (CD19 + /CD3 -), CD4 + T cells (CD3 + /CD4 + ), CD8 + cytotoxic T cells (CD3 + /CD8 + ), γδT cells (CD3 + /TCR + ), macrophages and dendritic cells (CD11b + /CD11c + ) will be quantitatively determined by flowcytometry. Lung sections will be stained with labeled antibodies (Table 2) as described [7] for visual identification of varying classes of immune cells. Adaptive immune responses against AdE will be characterized by neutralization antibody assay as described [4] except human 293 cells will be There will be 13 young (2-3 months old) female mice per group; survival rates will be determined and body weights will be taken on a daily basis for 20 days post-IFV challenge; body-weight loss of > = 30% will be taken as the disease endpoint; A195 buffer, AdE, PR8, CA04 will be prepared as described [1] except AdE will be mass-produced in human 293 cells; statistical analysis will be performed as that described in Fig 1 legend. Three mice in each group will be euthanized 3 days post-IFV challenge, with their lungs resected for determining IFV titers by plaque assay in MDCK cells in conjunction with lung histopathology assay as described [1]. https://doi.org/10.1371/journal.pone.0241266.t001
In Stage II studies, we will also test the hypothesis by mitigating the hypothetical pro-IFV response induced by IFV infection with zanamivir (Relenza) and/or a variety of anti-inflammatory agents in an attempt to reset innate immunity within the airway post-IFV infection for allowing AdE to induce an anti-influenza state in a post-exposure-therapy manner. We will screen a variety of anti-inflammatory agents including aspirin [9], rapamycin [10], dexamethasone [11], etc. in a shotgun manner by intranasal instillation of an anti-inflammatory agent with or without zanamivir in conjunction with AdE administration at varying time points post-IFV challenge, and subsequently determine the agents' effects on influenza by taking daily body weights and determining survival rates of IFV-infected mice.

Ethics statement
Three biosafety protocols pertaining to coronavirus and IFV research have been approved by the University of North Texas Health Science Center (UNTHSC) Institutional Biosafety Committee (IBC) during 2019-2020; one animal-use-protocol application pertaining to these studies was approved by the University of North Texas Health Science Center Institutional Animal Care and Use Committee (UNTHSC IACUC) on October 8, 2020, with the approval number IACUC-2020-0022. The proposed mouse studies will be performed at the UNTHSC animal facility.

Cell culture supplies
Mass production of AdE, PR8, and CA04 is fundamental to this project. The planned experiments in this project require propagation of human 293 cells for AdE production; and Table 2. Antibodies for characterizing immune cells that infiltrate into the lung as a response to pulmonary infection by AdE and/or IFV.

Labeled antibody Mouse immune cells to be stained by the antibody
Anti-mouse CD3 All T cells We will characterize the panel of immune cells within the lung with a variety of labeled antibodies before and after intranasal instillation of AdE and/or IFV particles. Additional antibodies may be added to the list for expanding the scope as described [8]. https://doi.org/10.1371/journal.pone.0241266.t002 propagation of MDCK cells for PR8 and CA04 production. We are budgeting $4,000 for cell culture supplies.

Laboratory supplies and reagents
This budget includes HiTrap Capto Q columns for purification of viral particles, pipette tips, glassware, plasticware, cryovials and boxes, etc. We are asking for $1,000.

Animal costs
Animal studies will be performed within the animal facility at UNTHSC. Each inbred mouse costs approximately $25; and we will need approximately 400 mice with 5 mice per cage; mice will be maintained for approximately 30-70 days (a subset of mice is expected to be killed by influenza before the experiment is terminated). We are budgeting $20,000 for animal work (purchase and maintenance).