Viral Infectious Diseases

## **Chapter 6**

## Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients with Chronic Epstein-Barr Virus Infection

*Irina A. Rakityanskaya,Tatiana S. Ryabova and Anastasija A. Kalashnikova*

### **Abstract**

NK cells play an important role in combating viral infections. In this study, we examined the effect of therapy with recombinant interferon gamma (Ingaron) on cytotoxic activity of NK cells. Sixty patients with chronic Epstein-Barr virus infection (CEBVI) were examined. All patients were treated with Ingaron at a dose of 500,000 IU every other day IM. Initially, they received 10 injections of Ingaron followed by a 10-day break to assess the dynamics of clinical and laboratory parameters. Then, the treatment was continued with five injections of Ingaron. In total, each patient received 15 injections or a total dose of 7,500,000 IU. The administration of recombinant interferon gamma at a total dose of 5,000,000 IU stimulated spontaneous and induced degranulation of NK cells in patients with CEBVI. After a full course of 7,500,000 IU of recombinant interferon gamma, CD107a expression on NK cells decreased but remained higher than before the onset of therapy and exceeded reference values. Thus, the maximum activity of NK cells in the peripheral blood of patients with CEBVI was reached 10 days after the administration of Ingaron at a total dose of 5,000,000 IU.

**Keywords:** NK cells, cytotoxic activity, chronic Epstein-Barr virus infection, recombinant interferon gamma, therapy

### **1. Introduction**

### **1.1 Epstein-Barr virus**

The Epstein-Barr virus (EBV) is a lymphotropic herpesvirus type 4 and the causative agent of infectious mononucleosis [1, 2]. The virus was first discovered and isolated in cells from African Burkitt's lymphoma by Epstein M.A., Barr Y.M., and

Achong B.G. in 1964 and later it was found that EBV is widespread throughout the world [3]. The first identified variants of EBV were type 1 (type A) and type 2 (type B). Type 1 (B95-8, GD1, and Akata) is the main type of EBV prevalent worldwide and type 2 (AG876 and P3HR-1) is more common in Sub-Saharan Africa [4]. EBV variants have different replicative properties and a person can become superinfected with two or more strains.

EBV infects most people during their lifetime and, after the acute phase, persists until the end of a person's life. The life cycle of EBV is characteristic of a virus with a large DNA envelope, consisting of phases of primary infection, latency, and lytic reactivation. The EBV genome encodes nine different glycoproteins (GPS) for envelope entry. Currently, 13 GPS have been identified, 12 of which are only expressed during the productive cycle of lytic replication. One of which (BARF1, a decoy viral colony-stimulating factor 1 receptor (vCSF1R)) can also be expressed during the latency period [5]. The tropism of newly released EBV virions is determined by the GPS envelope, which appears to differ depending on the host cell [6]. EBV infects B cells *via* the CD21 receptor, epithelial cells, and, less commonly, T or NK cells. Infection of B-lymphocytes leads to the preservation of the EBV genome as an episome.

The virus undergoes lytic replication in epithelial cells and establishes a lifelong latency in circulating memory B lymphocytes, periodically reactivating from latency [7]. Epithelial cells are the first to become infected, as EBV is transmitted to recipients *via* saliva. B cells become infected when EBV is released from the oropharyngeal epithelial cells [6, 8]. Lytic replication increases the pool of latently infected cells. EBV virions released from epithelial cells prefer B cells and EBV virions released from B cells prefer epithelial cells due to the composition of the GPS envelope [9]. EBV reactivation (lytic phase) under conditions of psychological stress leads to a weakening of cellular immunity and can stimulate EBV reactivation and replication by weakening the cellular immune system's control over viral latency. Chronic EBV reactivation is an important mechanism in the pathogenesis of many oncological and autoimmune diseases [10]. During the lytic phase, the full set of virus genes is expressed and a progeny virus is produced. Virions produced during lytic replication in epithelial cells replenish the viral reservoir in an infected individual and ensure the transmission of the virus in the population. During the latency period, the virus expresses only a limited number of genes necessary to maintain the viral genome (in the form of an episome in the nucleus) and evade the host's immune system [8].

### **1.2 Natural killer cells**

Natural killer cells (NK cells) are a unique subpopulation of cells that lack antigenspecific receptors. NK cells have high cytotoxic activity and produce a large amount of interferon gamma (IFN-γ) when they interact with transformed or infected target cells [11]. The recognition process of target cells consists of the signals they receive from activating and inhibitory receptors encoded by the germline. As a result of these interactions, the identification or death of target cells occurs.

In the absence of inhibiting signal, continuous stimulation of activation receptors deactivates NK cells and reduces their activity. When target cells transform or become infected, the expression of HLA Class I on their surface may cease. Therefore, multiple NK cell receptors along with the presence of activated cytokines and cells that adapt and express various receptors in NK cell compartment promote responsiveness of these innate cytotoxic lymphocytes [12].

*Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

The activity of NK cells is also regulated by four additional mechanisms:

1. repertoire of NK cells;

2.activation by cytokines or priming of NK cells;

3.adaptive or memory-like differentiation of NK cells; and

4.licensing of NK cells.

There are 30,000 subpopulations of NK cells that differ in respect of inhibiting and activating receptor expression.

During Epstein-Barr virus (EBV) infection, NK cell expansion occurs in peripheral blood, and the cytotoxicity of NK cells to EBV-infected cells increases. The expansion of early differentiated NK cells lasts for at least 6 months [13]; however, the cells in this period stop to proliferate and acquire CD57 marker of aging [14]. A higher count of NK cells correlates with a lower EBV titer in peripheral blood, which suggests that the level of NK cell response depends on the clinical severity of the disease. It was recently demonstrated that induction of lytic replication in EBV-infected B cells leads to an increased destruction of NK cells. This may suggest that EBV-infected cells become a target for NK cells. It is assumed that NK cells have no significant control over the establishment of latency. Therefore, although the population of NK cells increases and is capable to kill target cells, no influence on the viral load during lytic or latent infection is observed. It was shown that NK cells play a crucial role in the control of herpes virus infections when the presence of viral antigens leads to the activation, proliferation, and accumulation of these cells in sites of infection [15]. Therefore, NK cells are an important factor in the control of initial EBV infection because they eliminate infected B cells and enhance antigen-specific response of T cells by the release of immunomodulatory cytokines.

### **1.3 Antiviral functions of IFN-γ**

Currently, there are specific antiviral drugs, but there is no single approach to the treatment of chronic EBV infection. The antiherpetic drug must specifically inhibit the replication of the virus. The moment the virus evades the host's immune response, it is a potential target for chemotherapeutic effects. The higher the selectivity of the drug, the narrower the spectrum of its antiviral activity, since the drugs affect only the stages of virus replication. Drugs approved for the treatment of herpes simplex virus 1 (HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV), and human cytomegalovirus (HCMV) are nucleoside (i.e., acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), and its oral prodrugs; valacyclovir (VACV), famciclovir (FAM), and valganciclovir (VGCV), respectively), nucleotide (i.e., cidofovir (CDV)), and pyrophosphate (i.e., foscavir (foscarnet sodium), PFA) [16, 17]. None of these drugs have received FDA (Food and Drug Administration) or EMA (European Medicines Agency) approval for the treatment of EBV infections [8, 18].

IFN-γ has a direct antiviral action on infected cells, and also activates local dendritic cells, macrophages, and NK cells, modulates differentiation and maturing of T cells and B cells, and promotes inflammation and antiviral functions [19]. Suppression of any stage of the life cycle of virus can suppress the replication of its genome during infection. IFN-γ is a powerful antiviral cytokine that disrupts the life cycle of virus in stimulated cells on various stages. There are several mechanisms of its action:


Some well-known antiviral functions of IFN-γ lack specific antiviral mechanism. For instance, IFN-γ strongly induces indoleamine-2,3-dioxygenase (IDO) and nitric oxide synthase (NOS). The depletion of tryptophan and the production of nitric oxide (NO) due to the expression of IDO and NOS have pronounced antiviral effects, but their molecular details generally remain unclear. IFN-γ can also manifest noncytolytic activity against some viruses. However, specific targets and effector proteins of IFN-γ-dependent antiviral response are largely unknown [20]. Further studies are needed to clarify the antiviral mechanisms of IFN-γ, especially considering its strong immunomodulatory action.

In Russia, the only registered IFN-γ drug is Ingaron manufactured by OOO NPP FARMAKLON. It is obtained by the microbiological synthesis in recombinant *Escherichia coli* strain and purified by column chromatography. The molecule consists of 144 amino acid residues; the first three residues (Cys-Tyr-Cys) are replaced with Met.

**The objective** of the present research is to study the recombinant IFN-γ (Ingaron) action on dynamics of content of EBV DNA in the saliva sample, the killer cells content post-therapy, and changes of cytotoxic activity of the killer cells, and assess the influence of cytotoxic activity of the natural killers on the clinical complaint development and progression of illness in patients with CEBVI after the therapy completion.

### **2. Materials and methods**

*Patients*. The study group included 60 patients with CEBVI (39 women and 21 men; mean age 34.64 1.21 years). The duration of CEBVI was from first complaints to laboratory confirmation and the diagnosis was 2.85 0.56 years. Forty-three patients (71.66%) had frequent exacerbations of antibiotic-resistant chronic tonsillitis in childhood, and 15 patients (25%) had a history of acute infectious mononucleosis. All patients had a differential diagnosis of CEBVI versus other viral infections (human immunodeficiency virus, viral hepatitis, cytomegalovirus infection), toxoplasmosis, helminth infestations, and autoimmune diseases associated with EBV infection. The diagnosis was confirmed on a previous stage by laboratory investigation and expert examination, and the patients were referred for the immunological treatment. Those

### *Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

patients, who received antiviral and immunomodulatory therapy within the last 6 months, were not included in the study.

CEBVI characterizes with a prolonged treatment and frequent recurrences with clinical and laboratory signs of viral activity (mononucleosis-like symptoms) that are described in detail in the literature [21]. Patients suffer from low-grade fever (37.1— 37.3 °C), weakness, unmotivated tiredness, excessive sweat (especially at night), constant discomfort and/or pain in throat, lymphadenitis, swelling of the nasal mucosa with postnasal mucus drip, and stomatitis. Some patients have cough, skin eruptions, arthralgia, and muscle pain in body and limbs. Manifestation of conjunctivitis and otitis is possible. Neurological disorders such as headache, impaired memory and sleep, impaired concentration, irritability, tearfulness, and depressive tendencies may occur. Internal organs may increase in size (hepatomegaly and splenomegaly evidenced by ultrasound investigation) and a heavy feeling under the right ribs may be present. Some patients complain about frequent cold-related diseases and concurrent herpes virus infections. Many of these patients have a history of prolonged stress and psychoemotional and physical overload that exacerbates their condition.

This clinical study was performed in accordance with the World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects (2013); the protocol to the Convention of the Council of Europe on Human Rights and Biomedicine (1999); and Articles 20, 22, 23 of the Russian Federal Law no. 323-FZ on fundamental healthcare principles in the Russian Federation (November 21, 2011 as revised on May 26, 2021). The protocol was approved by the ethical committee of OOO Tsentr dializa Sankt-Peterburg, Fresenius Medical Care. All participants signed a voluntary informed consent. Patients included into the study had no other diagnosed infections, chronical diseases, or changed immune status that could affect the results.

*Clinical methods* included taking of history, data on previous treatment, and concurrent diseases. The clinical condition of patients was assessed traditionally with consideration of objective data and complaints at the time of examination registered using a three-point scale (0—no symptoms, 1—mild symptoms, 2—moderate symptoms, and 3—severe symptoms).

*Treatment schedule.* All patients received therapy with intramuscular recombinant IFN-γ (Ingaron) at a dose of 500,000 IU every other day. The course consisted of 15 injections. In the first phase, patients received 10 injections (5,000,000 IU) of Ingaron at a single dose of 500,000 IU followed by a 10-day break to assess the dynamics of clinical and laboratory parameters. In total, 500,000 units are the standard daily dose of the drug, which is recommended by the manufacturer. After that, the therapy was resumed and patients received five injections (2,500,000 IU) of Ingaron. Ten days after the last injection, the examination was repeated. In total, every patient received 15 injections (7,500,000 IU) of Ingaron (see **Figure 1**).

**Figure 1.** *Treatment regimen.*

All patients tolerated the drug fairly well. After the first 3–5 injections, 14 patients (23.33%) had a fever (37.3–37.5°C), myalgia, chills, sore throat, and increased postnasal drip. This was considered an exacerbation of CEBVI in association with the drug. After the seventh and eighth injections, these complaints fully disappeared.

### **3. Methods of examination using real-time polymerase chain reaction (PCR) with fluorescence hybridization**

Viral DNA was detected in saliva samples using real-time polymerase chain reaction (PCR) with fluorescence hybridization, AmpliSens EBV/CMV/HHV6-screen-FL kits by the Central Research Institute of Epidemiology (Russia) were used. The unit of measurement used to estimate the viral load during DNA extraction from saliva is the number of copies of EBV DNA per ml of sample. According to the instructions, this indicator is calculated using the formula: Number of DNA copies = CDNA x 100, where CDNA is the number of copies of the viral DNA in the sample. The analytical sensitivity of the test system is 400 copies/ml.

*Cytotoxic activity of killer cells* was evaluated based on the spontaneous and induced expression of CD107a (LAMP, lysosomal-associated membrane protein on the cell membrane of lymphocytes, which is a sign of degranulation of lysosomes). CD107a was assessed after co-culture of peripheral blood mononuclear cell (PBMC) with target cells (K562, chronic human erythromyelosis). K562 cells express a range of ligands (MICA, MICB, ULBP2, and ULBP4) for NKG2D receptor of cytotoxic lymphocytes. The interaction between NKG2D and the ligands leads to the degranulation of lysosomes in NK cells, TNK cells, and lymphokine-activated CD8+ T cells, and to the expression of CD107a on their membranes. Therefore, the test reveals the ability of killer cells to participate in NKG2D-dependent cytolysis of target cells. Blood was collected in a vacutainer with heparin lithium as an anticoagulant. Sample preparation included separation of mononuclear cells suspension from peripheral blood using density gradient with subsequent washing, co-culture of PBMC and K562 in 10:1 ratio in a CO2 incubator for 20 hours with anti-CD107a-AlexaFluor 647 monoclonal antibodies (BioLegend), and staining with anti-CD3-FITC/CD(CD16+56)-PE and anti-CD45PC5 monoclonal antibodies (Beckman Coulter). To assess the spontaneous cytotoxic activity, a respective volume of RPMI medium (Biolot) was added to PBMC suspension instead of K562. The samples were analyzed using a Navios flow cytometer (Beckman Coulter) up to 1,000 events in a minimum subpopulation of NK or TNK cells. The population of lymphocytes was defined as CD45+brightSSdim. The relative number of cells with CD107a expression (CD107a+) was assessed in subpopulations of NK, TNK, and T lymphocytes. The stimulation index was calculated as a ratio of induced expression to spontaneous expression of CD107a.

*To assess the relative number of NK cells,* multicolor flow cytometry was applied during the study of lymphocyte subpopulations in peripheral blood collected from the ulnar vena in vacutainers with EDTA. The samples were prepared according to the manufacturer's protocol. The following monoclonal antibodies were used: anti-HLADR-FITC, anti-CD4-PE, anti-CD3-ECD, anti-CD56-PC5.5, anti-CD25-PC7, anti-CD8-APC, anti-CD19-APC-AF700, and anti-CD45-APC-AF750. VersaLyse was chosen for the lysis of red blood cells. The samples were analyzed using Navios flow cytometer and respective reagents (Beckman Coulter) up to 5,000 events from the CD45+brightSSdim lymphocytic region. NK cells were defined as

*Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

CD3CD56+ CD45+brightSSdim events. The absolute number of NK cells was calculated from the results of clinical blood analysis.

*Statistical analysis.* IBM SPSS Statistics ver. 26 software package (Armonk, NY: IBM Corp.) was used for statistical analysis of the data. Group results were presented as the mean (M) standard deviation (SD). Statistical comparison between groups of patients was performed using nonparametric Mann—Whitney U test. Differences in continuous variables were assessed using independent samples Student's t-test and were considered statistically significant if p ≤ 0.05. Parametric (Pearson correlation) and nonparametric (Spearman's rank, Kendall's tau) methods were also applied. To check the independence of observations, linear regression analysis with the coefficient of determination (R2 ), Durbin—Watson statistic, and analysis of variance (ANOVA) were applied. Fisher's exact test (F) was calculated to check the statistical significance of the model. A standard β coefficient with 95% confidence intervals was calculated. The threshold significance of differences in this study was 0.05.

### **4. Results**

### **4.1 The effectiveness of treatment with recombinant IFN-γ (Ingaron)**

In all patients (n=60), EBV infection was confirmed by PCR reaction in saliva samples. The study of DNA PCR was carried out 10 days after the administration of 10 injections of Ingaron (total 5,000,000 IU). After that, patients received five more injections of Ingaron (2,500,000 IU), and the number of copies of EBV DNA in saliva samples was assessed by PCR again. The results are shown in **Table 1**.

The data show a significant decrease in the number of EBV DNA copies in saliva samples 10 days after a course of 10 injections (5,000,000 IU) of Ingaron; 21.66% of patients had a negative result of PCR test. After a full course of 15 injections (7,500,000 IU) of Ingaron, 31.66% of patients had a negative result of PCR test of saliva samples (**Figure 2**). This means that the effectiveness of antiviral therapy confirmed by negative PCR was significantly higher after 15 injections than after 10 injections (p = 0.001).

### **4.2 Presence of NK cells in peripheral blood**

The presence of NK cells in peripheral blood was assessed before treatment, after 10 injections, and after 15 injections of Ingaron. The results are shown in **Table 2** and **Figure 3**.


**Table 1.**

*The dynamics of the number of copies of EBV DNA after treatment with Ingaron in patients with CEBVI.*

### **Figure 2.**

*The dynamics of EBV DNA in saliva samples before and after treatment with Ingaron in patients with CEBVI.*


### **Table 2.**

*The content of NK cells (%) in blood before and after the treatment with Ingaron in patients with CEBVI.*

### **Figure 3.**

*Dynamics of the content of NK-cells (%) in the blood before and after treatment with Ingaron in patients with CHEBVI.*

*Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

The data show that the presence of NK cells in peripheral blood is significantly higher after administration of 10 injections of the drug and decreases after 15 injections, but generally still exceeds the level before treatment.

### **4.3 Dynamics of cytotoxic activity of NK cells**

Next, the dynamics of cytotoxic activity of NK cells before treatment and 10 days after 10 injections of Ingaron was assessed (**Table 2**). The expression of CD107a on NK cells 10 days after 10 injections of Ingaron significantly increased and exceeded referent values. This means that the introduction of recombinant IFN-γ at a total dose of 5,000,000 IU stimulates spontaneous and induced degranulation of NK cells and stimulation index in patients with CEBVI. After a full course of treatment (7,500,000 IU of recombinant IFN-γ), the expression of CD107a on NK cells reduced but was still higher than before treatment and exceeded referent values. Therefore, the maximum activity of NK cells in peripheral blood in patients with CEBVI was observed 10 days after administration of a total dose of 5,000,000 IU Ingaron (**Table 3**).


#### **Table 3.**

*The dynamics of the expression degranulation marker CD107a on NK cells, before treatment, and 10 days after 10 injections of Ingaron in patients with CEBVI.*


#### **Table 4.**

*The dynamics of the expression degranulation marker CD107a on NK cells, before treatment and 10 days after 15 injections of Ingaron in patients with CEBVI.*

Next, the dynamics of cytotoxic activity of NK cells 10 days after 15 injections of Ingaron was analyzed (**Table 4**).

### The data from **Table 4** are shown in **Figure 4**.

The dynamics of the content of NK cells and cytotoxic activity visually resemble the sign "bell" or "arch" (∩) of varying severity. This direction of the obtained results indicates the development of a hyporeactive state of cells against the background of a longer administration of Ingaron (15 injections). The hyporeactive state of NK cells is a consequence of a decrease in the number of EBV DNA copies, which in turn is accompanied by a positive dynamics of clinical complaints after a full course of therapy (7.500.000 IU).

### **4.4 Dynamics of clinical complaints**

The next stage of the work was an analysis of the frequency of the main clinical complaints in patients before treatment and after 10 and 15 injections of Ingaron. **Table 5** and **Figure 5** show the dynamics of clinical complaints during therapy.


*Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

### **Table 5.**

*The frequency (%) of main clinical complaints before treatment and after 10 and 15 injections of recombinant IFN-γ in patients with CEBVI.*

The data show that after the introduction of 10 injections of ingaron, there is a significant decrease in the frequency of subfebrile temperature, sore throat, weakness, and manifestations of stomatitis. After the introduction of 15 injections of ingaron, the dynamics of clinical complaints are more evident: a decrease in the frequency of subfebrile temperature, sore throat, weakness, chills, stomatitis, and swelling of the nasal mucosa with postnasal mucus drip.

### **Figure 4.**

*The dynamics of the expression of CD107a marker of degranulation of cytotoxic granules by NK cells before and after 10 and 15 injections of Ingaron in patients with CEBVI.*

### **Figure 5.**

*The frequency (%) of main clinical complaints before treatment and after 10 and 15 injections of recombinant IFN-γ in patients with CEBVI.*

*Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

### **4.5 Prognostic value of the presence of CD3CD16+CD56+ cells in peripheral blood**

To reveal the prognostic value of NK cells, linear regression analysis was performed with coefficients of determination (R2 ) calculated using Durbin—Watson statistic, and also the analysis of variance (ANOVA), Fischer exact test (F), and standard beta coefficient (β) with 95% confidence interval. The results of the criterion F and the coefficient ß, indicating the significance of the obtained regression models, are presented below:


The results of linear regression show that the presence of CD3CD16+CD56+ subpopulation of cells in blood before treatment is a predictor of the development and progression of clinical complaints in patients with CEBVI.

### **5. Resume**

NK cells play a critical role in fighting EBV infection. NK cells are cytotoxic to EBV-transformed cells during the acute phase and limit the EBV viral load [22]. The mechanism of action of NK cells against EBV is not well understood. NK cell cytotoxicity is strongly activated by EBV-induced ligands on infected B cells. Activated NK cells use three main strategies to kill virus-infected cells:


NK cells can prevent EBV entry into B cells and prevent B cell transformation via IFN-γ [23]. Human peripheral blood NK cells recognize EBV-replicating B cells by suppressing MHC class I surface molecules on infected cells [24].

The human NK cell compartment has up to 30,000 different subpopulations. Human herpesviruses promote the expansion of distinct subpopulations of NK cells, which then persist at an increased frequency for several months after infection.

During this time, they stop proliferating and acquire the aging marker CD57. Uncontrolled EBV infection develops with a decrease in NK cell compartments [25]. The hallmark of NK cell activation is degranulation, that is, the release of the contents of lytic granules. The granules consist of secretory lysosomes containing a dense core, various proteins, and take part in cytotoxic functions (e.g., perforin, granzymes) on the surface of the target cell. The inner surface of the granules is covered with CD107a (lysosome-associated membrane protein 1), a highly glycosylated protein that appears on the cell surface due to the fusion of lysosomes with the plasma membrane. Degranulation leads to the expression of CD107a on the cell surface and depletion of intracellular perforin. After degranulation, CD107a is exposed on the surface of the cytotoxic lymphocyte, protecting the membrane from perforin-mediated damage [26]. Resting NK cells, upon receiving signals for degranulation, are able to express surface CD107a and mediate cytotoxicity. Polarization and degranulation of cytolytic granules are two steps in NK cell cytotoxicity that are controlled by separate signals from different receptors. Neither polarization nor degranulation is sufficient for the efficient lysis of target cells. The ability of NK cells to kill virus-infected cells occurs before the "depletion" of NK cells, which is probably due to the depletion of cytolytic granules. The results of the NK cell degranulation analysis have been shown to correlate with standard cytotoxicity results. That is, CD107a expression may be a sensitive marker for determining cytotoxic activity [27].

In our study, the expression of CD107a degranulation marker on NK cells 10 days after the administration of 5,000,000 IU Ingaron significantly increased and excessed reference values. This means that the introduction of recombinant IFN-γ at a total dose of 5,000,000 IU stimulates spontaneous and induced degranulation of NK cells in patients with CEBVI. After the full course of treatment with 7,500,000 IU of recombinant IFN-γ, the expression of CD107a on NK cells decreased but was still higher than before the treatment and exceeded reference values. The maximum activity of NK cells in the peripheral blood of patients with CEBVI was achieved 10 days after the administration of a total dose of 5,000,000 IU Ingaron. Therefore, the results of the analysis of NK cells degranulation correlate with standard results on cytotoxicity as shown in studies by Alter G. et al. [27]. The expression of CD107a can therefore be a sensitive marker of cytotoxic activity of NK cells. The maximum expansion of NK cells in the peripheral blood of patients with CEBVI was observed after the administration of a total dose of 5,000,000 IU Ingaron, after additional five injections (2,500,000 IU) Ingaron, that is, after a full course of 7,500,000 IU Ingaron, the content of NK-cells decreased, but did not reach the initial level. The dynamics content and cytotoxic activity of NK cells visually resemble the sign "bell" or "arch" (∩) of a different curvature. In 1985, Talmadge, J. E. et al. were the first to demonstrate the bell-like curve of the dependency of NK cells presence on the dose of recombinant IFN-γ *in vitro* and *in vivo* [28]. They experimented on mice and showed that the activity of NK cells sharply increases 24 hours after the administration of recombinant IFN-γ and reaches a peak 48 hours after administration. The drug was several times more effective to increase cytotoxicity mediated by NK cells compared with IFN-α; its repeated administration led to a decrease in NK cells activity, and a hyporesponsive state developed. Preclinical and clinical studies of recombinant IFN-γ also showed a bell-like dependency on the dose when NK cells were induced by multiple or high doses of the drug [29]. This systemic hyporeactive state occurs not only in the spleen and peripheral blood, but also in NK cells isolated from the lungs and liver. In this case, the hyporesponsiveness of NK cells occurred when normal cells stimulated NK cells but the inhibiting signals from HLA Class I molecules were absent, *Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

or when excessive stimulation was stronger than inhibiting signals. Constant engagement of activating receptors and the lack of inhibiting receptors led to the hyporesponsiveness of NK cells.

Experiments on mice showed that the constant interaction of the activating Ly49H receptor with NK cells leads to the development of hyporeactivity of NK cells due to changes in the downstream signaling pathways from the receptor to the adapter molecule. The constant interaction of Ly49H receptor with its ligand *in vivo* results in a weak response of Ly49H+ NK cells to further stimulation from other receptors, whereas Ly49HNK cells remain unaffected. Hyporesponsiveness of NK cells correlates with the suppression of the activity of Ly49H receptor on the cell membrane. When effective inhibiting signals are absent, NK cells experience sustained activation and become hyporeactive, which is known as the "disarming" model [30]. However, the most important mechanisms that lead to the hyporesponsiveness of NK cells need further investigation.

Based on the previously published results of studies on the mechanism of development of NK cell hyporeactivity and our data, it becomes obvious that long-term administration of recombinant interferon-γ in patients with chronic EBV infection leads to the development of a decrease in the function of NK cells. In our study, the development of a hyporeactive state of NK cells against the background of a longer administration of ingaron (15 injections) is accompanied by a decrease in the number of copies of EBV DNA in saliva samples and a more pronounced positive dynamics of clinical complaints in patients after a full course of therapy (7.500.000 IU).

The study of the inhibitory effect of pure recombinant human (rh) IFN-α and IFN-γ on EBV infection began in the late 80s and early 90s of the twentieth century. In 1986, Shigeo Kure et al. demonstrated that none of the rhIFNs lack pronounced inhibiting effect on EBNA expression in hidden EBV-infected Raji and Daudi cells. These results suggest that rhIFN act mostly on the early stage of EBV infection [31]. It was demonstrated in an experimental setting that pretreatment of Vero cells with either IFN-β or IFN-γ inhibits HSV-1 replication by less than 20-fold. Сo-treatment with IFN-β and IFNγ inhibits HSV-1 replication about 1,000 times [32, 33]. The authors proposed that a high level of inhibition after the introduction of exogenous IFN-γ was a result of a synergic interaction with endogenous IFN-α/IFN-β produced locally in response to HSV-1 infection. A study of the influence of purified recombinant interferons of all three classes on EBV-induced proliferation of B cells and immunoglobulin secretion showed that IFN-γ reduces B cell proliferation and immunoglobulin production if added 3–4 days after infection and that IFN-α and IFN-β effectively influence cell proliferation only within 24 hours. The authors showed that the antiviral effect of IFN-γ on EBV-infected cells is 7–10 times stronger than that of IFN-α and IFN-β [34, 35]. Our study demonstrated a significant decrease in the number of copies of EBV DNA in saliva samples 10 days after the administration of 5,000,000 IU of Ingaron, and the results of PCR test were negative in 21.66% of patients. After a full course of treatment with 7,500,000 IU Ingaron, 31.66% of patients had negative results of PCR test of saliva samples. This means that the full course of Ingaron is significantly more effective (p = 0.001). A strong and significant decrease in clinical complaints of patients was achieved after the full course of treatment.

### **6. Conclusions**

1. Ingaron is a recombinant human INF-γ preparation. It has a pronounced antiviral effect, which is expressed in a significant decrease in the number of EBV DNA copies in patients with CEBVI.


## **7. Future research directions**

It is necessary to carry out further investigation of how Ingaron affects dynamics of content of other subpopulations of lymphocytes of peripheral blood in the course of treatment by the medication. Also seems to be interesting to study production of the anti-inflammatory cytokines (IL-1β, IL-6, and TNF-β) in the course of the Ingaron treatment.

Based on preliminary results of this study, we suppose that Ingaron possesses manifest anti-viral action and is one of the activators of immune response. The medication can be used as a combination therapy for chronic Epstein-Barr infection, which will save working population and reduce burden on the healthcare system.

## **Authors' contribution**

Conception and research design—Rakityanskaya I. A.; material gathering and processing—Rakityanskaya I. A., Ryabova T. S.; data analysis and interpretation— Rakityanskaya I. A., Ryabova T. S.; lab research—Kalashnikova A.A.; statistical processing of data—Rakityanskaya I. A.; script composition—Rakityanskaya I. A., Ryabova T. S.; editing—Ryabova T. S.Kalashnikova A.A.; research supervision— Rakityanskaya I.A.; text writing and editing—Rakityanskaya I. A., Ryabova T. S., Kalashnikova A.A.; responsibility for integrity of all article's parts—Rakityanskaya I. A.; script further revision for important intellectual content—Rakityanskaya I. A., Ryabova T. S., Kalashnikova A.A. All the authors have made a substantial contribution to this study and approved the final script version.

*Recombinant Interferon Gamma: Influence on the Cytotoxic Activity of NK Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.108207*

## **Conflict of interests**

The authors declare the absence of conflict of interests.

## **Data sharing policy**

The statistical code, dataset used in support of the findings of this study are included within the article.

## **Financing**

The study did not have sponsor's support.

## **Author details**

Irina A. Rakityanskaya<sup>1</sup> \*, Tatiana S. Ryabova1,2 and Anastasija A. Kalashnikova<sup>3</sup>

1 Department of Allergology, Immunology and Clinical Transfusiology, Municipal Outpatient Hospital, Saint Petersburg, Russia

2 S.M. Kirov Military Medical Academy, Saint Petersburg, Russia

3 A.M. Nikiforov Russian Center of Emergency and Radiation Medicine, EMERCOM of Russia, Saint Petersburg, Russia

\*Address all correspondence to: tat-akyla@inbox.ru

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 7**

## Airborne Transmission and Control of Influenza and Other Respiratory Pathogens

*Jacob Bueno de Mesquita*

### **Abstract**

Despite uncertainty about the specific transmission risk posed by airborne, spray-borne, and contact modes for influenza, SARS-CoV-2, and other respiratory viruses, there is evidence that airborne transmission via inhalation is important and often predominates. An early study of influenza transmission via airborne challenge quantified infectious doses as low as one influenza virion leading to illness characterized by cough and sore throat. Other studies that challenged via intranasal mucosal exposure observed high doses required for similarly symptomatic respiratory illnesses. Analysis of the Evaluating Modes of Influenza Transmission (EMIT) influenza human-challenge transmission trial—of 52 H3N2 inoculated viral donors and 75 sero-susceptible exposed individuals—quantifies airborne transmission and provides context and insight into methodology related to airborne transmission. Advances in aerosol sampling and epidemiologic studies examining the role of masking, and engineering-based air hygiene strategies provide a foundation for understanding risk and directions for new work.

**Keywords:** airborne infection, inhalation exposure, infectious aerosols, anisotropic

### **1. Introduction**

Seasonal and pandemic influenza remain global threats. Seasonal flu kills up to 650,000 people each year and pandemics have the potential to cause millions of deaths and disrupt societies. Despite surpassing the 100-year anniversary of the 1918–1919 global influenza pandemic with a death toll estimated at over 50 million, present-day non-pharmaceutical prevention strategies—including engineering controls like germicidal ultraviolet technology (GUV), filtration, and ventilation—remain inadequately used to quell seasonal influenza epidemics and emerging pandemics as demonstrated with ongoing epidemiologic waves of COVID-19. Stringent social isolation remains an effective approach over the centuries but may only achieve population-level compliance for short periods of time. Testing, vaccination, and therapies are helpful but have not been available at the outset of emerging pandemics, and face issues of waning effectiveness as pathogens evolve, and logistical and social issues related to rapid production and equitable dissemination. It is widely

appreciated that the quest for improved non-pharmaceutical controls and vaccines is dependent upon knowledge of influenza virus transmission via direct contact, large droplet spray, and aerosol inhalation and deposition along the respiratory tract. Increasing precision and confidence of quantified risks posed by airborne and other transmission modes support better design and evaluation of engineering controls and other strategies to reduce population spread. To rapidly identify airborne pathogens and continually update knowledge about airborne infection potential of evolving pathogens, there is a need for sentinel epidemiologic and bioaerosol sampling surveillance systems.

Influenza intervention trials showed that the use of hand hygiene and surgical masks to reduce contact and large droplet exposure resulted in only mild risk reduction among susceptible household contacts of influenza cases and may have facilitated more airborne transmission [1]. Human challenge studies have shown that infection initiated through aerosols, compared with nasal instillation [2, 3], required a lower dose and resulted in more severe disease. Inhalation of bioaerosols is likely important for other acute, viral, and respiratory infections and was convincingly implicated by airborne viral transport computational fluid dynamic models for a deadly SARS-coronavirus outbreak [4–6]. The capacity to directly measure the extent and intensity of transmission risk posed by bioaerosols represents uncertainty for which research is needed. Failure to quantify the contribution of exhaled bioaerosols impedes the advocacy for and effective use of control measures and facilitates population vulnerability during seasonal epidemics and pandemics.

William Wells described the quantum theory of airborne infection [7] whereby infection risk is described by exposure to infectious doses, or quanta (which is, more specifically, the dose that would infect 63% of those exposed), generated by infectious individuals over time. Studies quantifying influenza virus and SARS-CoV-2 virus shed into exhaled breath aerosols using a Gesundheit-II (G-II) bioaerosol sampler support an understanding of airborne contamination by infectious individuals, provides a way forward for precisely estimating airborne infection risk in terms of virions with infectious potential and genome copies measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) [8–11]. Human challenge transmission trials offer one way forward to quantifying the airborne transmission risk between infectious and susceptible individuals with known levels of inhalation exposure to exhaled breath, paired with measurements of viral load in exhaled breath. Real-world study in congregate settings where exposure may be unavoidable, including in healthcare or other public gathering places offers another approach that may provide more generalizable findings, yet may be more logistically challenging to achieve valid estimates of exposure [12]. Yet emerging genomic sequencing methods that can identify viral mutations shared between epidemiologically linked individuals can confirm transmission chains and may offer clues to the specific mode of transmission [13].

Reliable prediction of airborne risk informs disease control efforts by providing information about the relationship between various levels of exposure control via engineering controls—air disinfection by GUV, ventilation, and filtration—and reduce airborne transmission. This information is needed to inform public health approaches and infrastructure design to provide appropriate air hygiene for mitigating emerging pandemic viruses before effective vaccines and therapies become available. This chapter provides an overview of airborne viral infection dynamics and control with a focus on the scientific underpinnings required for future epidemiologic study designs under which longitudinal surveillance of contact networks

*Airborne Transmission and Control of Influenza and Other Respiratory Pathogens DOI: http://dx.doi.org/10.5772/intechopen.106446*

can revolutionize understanding of airborne infection transmission by pinpointing transmission routes and refining estimates of infection risk by airborne and other modes in indoor spaces. Results from this line of research provide key information for guiding the strategic use of prevention methods—especially air disinfection by GUV—to protect against seasonal epidemics and pandemics in shared air spaces and, in particular, among immunologically vulnerable populations.

### **2. Disease burden and public health impact**

The Forum of International Respiratory Societies emphasizes that acute respiratory infections are the greatest contributors to the global disease burden, responsible for 4 million deaths annually. CDC reports that influenza resulted in 9–36 million illnesses and up to 56,000 deaths each year since 2010 in the US, with annual estimated direct and indirect costs of \$87 billion [14]. Respiratory infections cost over \$15 billion annually in the UK [15]. Globally, seasonal influenza kills up to 675,000 people each year and influenza pandemics have the potential to cause millions of deaths and severe societal disruption. The health and economic burdens are amplified in developing nations with less access to health services [16]. The devastating loss of life, the morbidity and economic losses from COVID-19, trends in spillover of pathogenic avian influenza to humans with pandemic potential, and an increasingly interconnected world, all create an urgent case for improved prevention methods.

Prevention of these substantial population health threats cannot rely solely on vaccines, which are often poorly matched to rapidly evolving strains. During pandemic, the lag time in the production and dissemination of vaccines leads to widespread vulnerability and underscores the need for interventions based on viral exposure reduction to interrupt transmission. It is widely appreciated that the quest for improved non-pharmaceutical prevention methods—including reducing exposures through building-level air disinfection—or social distancing, and vaccines are dependent on understanding transmission risk via contact, large droplet spray, and fine-particle aerosol respiration (i.e., airborne) [17]. Vaccine development benefits from an understanding of host-pathogen dynamics related to transmission mode. There is strong evidence supporting the critical role of airborne transmission, and it is well-recognized that infection initiated by the airborne route is likely to cause more severe symptoms compared to infections initiated by contact or large droplet spray [2, 3].

The US CDC typically has recommended protective behaviors such as washing hands, covering coughs, and donning masks to reduce contact and droplet exposure, but has provided little specific guidance related to air cleaning or respiratory protection (e.g., fit tested N95s) to mitigate aerosols that are capable of penetrating and circumventing surgical or cloth masks. Intervention trials showed that the use of hand hygiene and surgical masks to hinder contact and droplet exposure resulted in only mild risk reduction among susceptible household contacts of nearly 800 influenza cases and may have promoted a greater proportion of airborne transmission [1]. Furthermore, those most likely to be exposed to airborne influenza, due to the use of hand hygiene plus surgical mask, tended to present with more severe symptoms characterized by fever and cough. Despite some delays in intervention initiation and imperfect adherence, such trial conditions reflect realistic population usage, while randomization and robust sensitivity analyses support internal validity to the extent possible. Although implementation of masking and engineering controls such as

GUV, filtration, and ventilation are well supported by existing evidence, the state of the science benefits from investigation to better quantify airborne transmission risk and the extent of the effectiveness of GUV, filtration, and ventilation.

A clear, dose-response relationship between dormitory rebreathed air fraction and likelihood of retrospective, self-reported acute respiratory infection (ARI) was observed in a study of 3,712 students in Tianjin, China [18]. A separate airborne infection risk model suggested that increased clean air supply could effectively control population spread of ARIs including influenza but may not have much effect on highly contagious infections like measles [19]. However, this study used estimated values of influenza contagiousness based on an airplane outbreak [20], where there was uncertainty about outdoor air exchange, and all secondary influenza cases were assumed to be connected to the index. More recently a comparison of two university dormitories in Maryland, USA showed that compared with a dormitory with higher ventilation the dormitory with low ventilation had 4 times (95% confidence 0.69–163.02) the ARI rate, although the sample size of infections reported in the high ventilated dormitory reduced the ability to make more conclusive comparisons [21].

While modulating airflow and ventilation can influence airborne contamination quantities and human exposure, unequivocal evidence from exposure chambers demonstrates the inactivation of aerosolized respiratory pathogens including influenza [22], vaccinia virus [23], and TB [24] under exposure to upper-room 254 nm UV-C light (GUV), representing a highly effective strategy to interrupt airborne transmission. But whereas current control techniques are unlikely to be strategically deployed, improved characterization of risk by transmission mode enables the most effective use of existing control strategies and may provide health benefits knowledge to help catalyze investment by communities, government, and public health agencies.

### **3. The human-challenge transmission trial for quantifying infection modes**

A meeting of globally recognized influenza transmission experts was convened by CDC in 2010 to address knowledge gaps about the relative importance of influenza transmission modes that are reflected in uncertainty about hospital care and general population prevention guidelines [25]. The meeting discussed possible animal and human transmission experiments and explored the possibilities of conducting epidemiological studies with engineering and/or personal protective interventions. Although there was great enthusiasm for studies of population infection surveillance with upper room GUV or other airborne control interventions, preliminary work in this area was lacking. Ultimately it was determined that a human challengetransmission study with interventions to control for transmission mode, surveillance of aerosol shedding, environmental conditions, comparison of aerosol infectivity of experimental and naturally infected influenza cases would represent the most scientifically sound approach.

### **4. Aerobiologic pathway for influenza and other respiratory infections**

An abundance of laboratory evidence substantiates the aerobiologic pathway for influenza and other ARIs and supports new epidemiologic studies of transmission. The aerobiologic pathway [26], consists of a) generation of particles containing

### *Airborne Transmission and Control of Influenza and Other Respiratory Pathogens DOI: http://dx.doi.org/10.5772/intechopen.106446*

infectious microbes from the respiratory tract or environmental sources, b) maintenance of infectivity and persistence in the air before reaching a susceptible host, and c) deposition in at least one vulnerable locus in the respiratory tract of the new host.

With respect to infectious particle generation, exhaled breath particles contain a respiratory fluid lining of the small airways and are generated by small airway closure and reopening [27–29]. A team led by Milton at the University of Maryland observed 218 half-hour exhaled breath samples from 142 symptomatic influenza cases and detected culturable influenza virus in 39% of fine-particle aerosols (≤5 μm) with geometric means of 37 infectious particles by fluorescent focus assay and 3.8x104 RNA copies by qRT-PCR (geometric standard deviations 4.4 and 13, respectively) [11]. Using a G-II bioaerosol collection device to sample natural breathing (including incidental coughs), this research clearly shows that influenza cases can generate many virus-laden particles. The same research team using a similar methodology detected SARS-CoV-2 in 36% of fine and 26% of coarse aerosols, while also detecting infectious viruses [10]. Others using the G-II showed that singing produced the highest proportion of positive fine aerosols, followed by talking and breathing [30].

Once generated, infectious aerosols maintain infectivity and persist in the air before reaching a susceptible host. The airborne movement of infectious particles has been implicated in human and animal transmission of influenza and other respiratory pathogens. Computational fluid dynamics and multi-zone models simulating a threedimensional aerosol plume rising upwards and around an apartment building with a SARS-coronavirus index case predicted the location of secondary cases [4]. Noti and colleagues measured infectious influenza in aerosols that had traveled across a room [31]. Upward dispersion of aerosols with slow settling velocity has been confirmed by influenza. A transmission between infected guinea pigs housed >100cm below exposed animals [32]. Numerous ferret studies report similar results. The ability for airborne particles to travel and initiate disease was implied by two postal workers who became infected with Anthrax following a known release of spores and no other known exposures [33].

Biologically active airborne particles carry public health significance given the potential for prolonged suspension and scenarios of exposure before removal occurs or through recirculated air that has not been filtered or sterilized. Studies of biological decay in aerosolized virus maintained in a rotating drum demonstrated infectious potential for influenza [34] and coronavirus [35] after 23 hours and 6 days, respectively. Although the exact sizes of the laboratory-generated aerosols used were not reported, these studies demonstrate prolonged infectiousness in particles <10 μm. The rate of biological decay as a function of temperature and relative humidity has been characterized through laboratory manipulation of viral-laden droplets [36]; and through airborne simulations with bacteriophage Phi6, a surrogate for influenza and coronaviruses [37]. Reduced decay corresponded with lower droplet salt concentrations associated with high and low vapor pressures, consistent with epidemiologic observation of peak transmission during the hot and rainy season in the tropics, and the cold and dry season in temperate climates. However other research using aerosolized virus from human airway epithelial fluid suggests that the influenza virus remains infectious independent of relative humility [38]. This latter work may be more convincing given the use of a more realistic human model. The aerosol half-life of SARS-CoV-2 has been reported at 1.1 hours (95% CI 0.64–2.64) [39], with infectivity measured at 16 hours with potential for longer persistence under longer observation [40].

Inhalation of airborne virus and deposition at a vulnerable locus in the respiratory tract can initiate infection. A human challenge study demonstrated an infectious dose for inhaled influenza A aerosols as low as 0.6–3 TCID50 [2]. A study of exhaled breath from confirmed influenza cases showed that 99 and 87% of particles were less than 5 and 1 μm, respectively [28]. This shows that exhaled breath aerosols are well within the size range to penetrate the lower lung. Fine particle aerosols exhaled from naturally infected influenza cases have been shown to carry infectious viruses [9, 11]. Given that, epidemiologic, laboratory, and challenge studies fail to definitively confirm human airborne transmission and produce valid risk models, there is a need for methods that maximize external validity to community settings and enable confirmation of transmission modes for a range of ARIs. Observation of community transmission provides an ideal platform to validate risk models that parameterize the aforementioned aerobiologic path—viral aerosol generation, persistence, and deposition—leading to valid estimation of infectious dose. Observation of exposed, asymptomatic individuals satisfies the concerns of Fraser and colleagues, which identified asymptomatic cases as key to pushing R0 above one [41].

### **5. Studies of influenza transmission risk by mode and the anisotropic hypothesis**

Hand hygiene and face masks have been assessed for their potential to reduce influenza transmission and gain information about transmission mode-related risk. Cluster-randomized trials with hand hygiene and facemask interventions found mild reductions in risk among intervention users (effect for hand hygiene and facemask groups, separately) that did not reach statistical significance [42]. This finding was consistent with those from studies performed in Hong Kong and Bangkok that showed the effect of hand hygiene plus facemask to be small at best [1, 43, 44]. A similar result was observed for crowded, urban households in upper Manhattan after 19 months of follow-up in 509 households [45]. However, a meta-analysis showed that hand hygiene plus facemask interventions were associated with a statistically significant 27% reduction in transmission risk [46]. Hand hygiene alone had no significant effect but showed a trend toward reducing risk under higher humidity and suggesting a predominance of aerosol transmission in temperate climates that is weakened in tropical climates. Given that facemasks have been assessed to reduce viral RNA copies contained in coarse aerosols by 25-fold and fine aerosols by 2.8-fold [9], if such reductions are associated with reduced transmission risk, then the meta-analysis findings make sense. Several other studies and review papers provide extensive evidence for the role of airborne particles in both influenza [5, 47, 48], and SARS-CoV-2 [49–55] transmission.

The hypothesis that influenza is anisotropic—that the route of transmission influences disease presentation [5]—is supported by early studies of human exposure to influenza contained in aerosols and nasal droplets [2, 3, 56], where aerosol exposure was more likely to result in influenza-like disease characterized by fever and cough, compared with nasal mucosa exposure representative of contact and droplet routes. The community-infected cases documented by Knight and colleagues exhibited similar symptomatology as Alford's infected volunteers, suggesting a natural tendency toward aerosol transmission. These findings were more recently borne out in ferrets where aerosol-infected animals not only presented with more severe symptoms but also shed more virus than their nasally-inoculated counterparts [57]. Similarly,

cynomolgus macaques exposed to SARS-CoV-2 via aerosols were more likely to experience fever and severe respiratory pathology compared with those exposed via intratracheal/intranasal drops, suggesting similar anisotropy [58].

### **6. Findings from EMIT human challenge transmission trial**

The human challenge-transmission trial (Evaluating Modes of Influenza Transmission [EMIT], ClinicalTrials.gov number NCT01710111) was designed to achieve an expected 40% SAR, however, achieved an actual SAR of 1.3% [59]. This finding on its own fails to provide definitive results regarding transmission modes, yet the low transmission rate from close-quarters exposure of infectious influenza cases over four consecutive 12–16-hour days with sero-susceptible individuals suggests that the contact and spray-borne transmission modes were not important contributors. Comparison of this result with the proof-of-concept study that achieved a SAR of 8.3% under much lower exposure time and ventilation motivates discussion about the role of ventilation and exposure to airborne pathogens [60].

Bueno de Mesquita and colleagues used CO2 data from the transmission trial, and knowledge of aerosol viral shedding by experimentally infected primary cases (known as "viral Donors") and applied the rebreathed-air equation—a modification of the Wells-Riley equation—to estimate an infectious quanta generation rate and RNA copy number per infectious quantum [19]. This analysis showed that the particular group of exposed individuals where the single secondary infection was observed was among the group with the highest exposure to virus contained in the exhaled breath of the Donors to which they were exposed. This suggests that the transmission may have occurred through the airborne mode. Assuming this, the airborne quanta generation rate (*q*) (95% CI) for influenza in the controlled human transmission trial environment among infected Donors and airborne viral shedding Donors was estimated to be 0.029 (95% CI 0.0270, 0.03) and 0.11 (0.088, 0.12) per hour, respectively. The number of RNA copies per infectious quantum was 1.4E+5 (95% CI 9.9E+4, 1.8E+4). Given this quantum generation rate, and levels of viral shedding in a college campus community in dormitory rooms evaluated for exhaled breath exposure, the typical viral shedder presents a low risk of transmission to a susceptible roommate during three nights of exposure in a well-ventilated dormitory but a moderate risk in a poorly ventilated dormitory. Supershedders at the 90th percentile of fine aerosol shedding would present high risk even in the higher ventilated dorm. The effect of higher ventilation could be modeled using the rebreathed-air equation and typically points towards the need for levels of air exchange far beyond what might be achievable by ventilation alone, underscoring the importance of air disinfection by GUV and filtration to mitigate superspreading.

The next question is whether the EMIT human volunteers experimentally infected by intranasal droplets simulate naturally-acquired infections to a comparable degree. To address this question, the EMIT study included an investigation of community influenza cases presenting with influenza-like illness. There was a low probability artificial nasal inoculation would have resulted in the highest levels of symptom severity and viral shedding observed among naturally infected cases selected on the basis of febrile illness [61]. Findings from these analyses generate new knowledge about influenza infection, disease, and transmission and inform future studies aimed at improving our understanding of respiratory infection transmission dynamics and associated disease. There is limited data elsewhere about the extent of shedding as a

function of symptom profile, although asymptomatic individuals have been shown to shed 1–2 log10 RNA copies fewer than symptomatic influenza cases [62]. The extent to which asymptomatic infections may be more representative of populations infected by upper respiratory mucosal exposure is unclear.

The computed infectious quantum generation rate enables the comparison between estimated exposure to influenza virus and infection risk. Thus, given levels of exhaled breath aerosol viral shedding and ventilation rates for indoor shared air spaces, the Wells-Riley equation can be applied to estimate infection risk. Of course, this assumes that the assumptions inherent in the computation of the *q* in the EMIT human challenge-transmission trial can be generalized to other transmission scenarios. The population of susceptible volunteers had low HAI and MN titres, representing above-average susceptibility to the general population, suggesting *q* may be overestimated. The computed *q* must also be interpreted with caution because it represents a point estimate, with confidence bounds generated by empirical bootstrap, given that it was derived from a single transmission event. The *q* for influenza in the challenge trial is relatively low compared with the few estimations done for other respiratory infections. Yet applying the EMIT-derived RNA copy to infectious quantum relationship to naturally infected influenza cases shedding the most virus among 142 mostly healthy young adults gave a *q* value of 630 [63]. Analysis of a super spreading event on an airplane suggested *q* or 100 for influenza virus, while *q* for rhinovirus has been estimated at 4 [19]. Careful epidemiologic investigation of an explosive measles outbreak in an elementary school showed *q* of over 5,000 [64]. Analysis of a SARS-CoV-2 outbreak in a poorly ventilated restaurant yielded an estimated *q* of about 80 [55], while a super spreading event at a choir practice led to an estimated *q* of over 900 [65].

## **7. EMIT trial limitations and questions for future work**

The EMIT challenge-transmission trial, like Alford's challenge study with aerosol viral exposure, used a population with low pre-existing antibodies to the challenge virus subtype. Thus, these studies are useful for demonstrating transmission dynamics with susceptible secondary cases but lack generalizability to the general population with varying levels of immunity. That only one transmission event was observed in a Control Recipient represents a major limitation, as the mode of transmission cannot be well deduced, and the risk ratio represents the lower bound for infection risk and lends uncertainty to the confidence bounds. Nonetheless, the EMIT analyses attempted to a) learn what was possible about influenza transmission given that the study was unique in its design and the largest human-transmission trial conducted to date, and b) fully assess the limitations of the study design to inform future investigations. Numerous questions exist to drive future studies aiming to refine risk assessment and optimize population prevention strategies. Such questions include:


setting (e.g., dormitories, military barracks, nursing homes, hospitals, schools, occupational settings)?


### **8. Implications for study design**

Findings from the experimental challenge-transmission model should be evaluated in studies of real-world epidemiology and population transmission dynamics. This way the potentially important contributions of other important variables can be assessed: a) immunity and shedding dynamics, b) socio-behavioral factors related to human-human interaction and exposure, c) overall well-being including psychological stress, sleep, physical activity, and diet [66], d) features of the built environment where exposure occurs including temperature, humidity, sanitary ventilation (combination of outdoor airflow, filtration, and GUV), and e) the role of other airborne exposures including particulate matter, ozone, and nitrogen oxides.

The advantage of the experimental trial in a controlled environment is that a relationship can be drawn between viral shedding quantity and subsequent secondary attack rates, giving a dose-response relationship. However recent advances in genomic sequencing and bioinformatics show a path forward for using molecular markers, in combination with epidemiological contact and exposure surveillance, to confirm transmission chains [67–69]. Sequences from identified transmission pairs may be able to give information about infection mode if viral communities evolve distinctly in the lung versus the upper respiratory mucosa. There is evidence that influenza may manifest as compartmentalized infections in the lung and nasopharynx [11, 70]. Airborne transmission likely involves viral communities produced in the lung, while contact transmission likely involves nasal communities, thus enabling a path to identify infection route that requires characterization. Considering the nasopharynx and

lung as separate entities that carry the ability to infect independently, reconstruction of transmission chains in observed contact networks may be possible by analyzing shared variants [13]. Bayesian approaches can be used to infer transmission events for outbreaks that are not completely sampled and/or are ongoing [71].

A study that simultaneously monitored ventilation rates in two neighboring dormitories housing first and second-year students and respiratory infections among the population found a trend towards a higher infection rate in the dormitory with lower ventilation, suggestive of a relationship between inhalation exposure and infection risk [21]. This study also showed a gradient of exposure levels to exhaled breath between rooms in a corridor that could support epidemiologic investigation of transmission chains. Longer studies with larger populations should be done that combine contact investigation and sequence analysis to confirm transmission chains. Symptom assessment, specimen collection for quantification of mucosal and exhaled breath viral load, viral community, immune biomarkers, and other health-related factors related to stress would provide necessary data sources to assess the relationship between exposure and infection risk that could be modified by immunologic factors.

### **9. Conclusions and implications for public health practice**

Although new studies are needed to refine estimates of transmission risk by various modes to understand the relationships between infection mode, dose, symptoms, age, sex, immunity, and environment, the existing state of knowledge is sufficient to support the scientific underpinnings of public health interventions aimed at reducing transmission and population epidemics through targeted airborne exposure control. At the very least, airborne infection preventive measures should be used as part of precautionary strategies to protect populations from loss of life and livelihood associated with emerging pandemics. In the case of influenza, it may be that the infectious generation rate of the average infectious aerosol shedder is low enough to pose an only mild risk under conditions with abundant sanitary ventilation but may pose moderate to severe risk under conditions of less sanitary ventilation (**Figure 1**). Fine particle aerosol supershedders may pose a substantial risk regardless of sanitary ventilation. Although they may be quite rare in the population, supershedders may account for most of the population spread as shown in the case of SARS-CoV-2 [72, 73]. Investigation of exactly how much of this risk can be attenuated by engineering controls opens the door for well-informed exploration of building design and operation strategies. That sanitary ventilation measures provide contribute to the control of any airborne transmitted pathogen represents a major advantage.

The magnitude of infection control measures required to prevent the community spread of airborne contagion is related to the infectivity of the pathogen (i.e., infectious dose shedding rate). Testing, quarantine, and isolation are critical measures to interrupt transmission by removing exposures to infectious sources and should always be considered. Yet there are challenges with achieving widespread access to sensitive tests for emerging pathogens, and compliance with quarantine and isolation procedures. Engineering controls including GUV, filtration, and ventilation provide an effective layer of protection that can be facilitated by the government as a social good requiring little if any behavior change or compliance at the population scale. Engineering controls—with an emphasis on GUV when dealing with highly infectious pathogens—can help move societies beyond reliance on social isolation and masking, especially given the social fatigue with these measures observed after more than two

*Airborne Transmission and Control of Influenza and Other Respiratory Pathogens DOI: http://dx.doi.org/10.5772/intechopen.106446*

#### **Figure 1.**

*Factors contributing to elevated risk of airborne infection transmission. Supershedders emitting high rates of infectious aerosol through exhaled breath or another aerosolization mechanism (e.g., aerosolization from toilet flush) can lead to high risk alone, or average spreaders can generate high risk under conditions of low sanitary ventilation or among populations of immunologically susceptible populations.*

years of COVID-19 pandemic [74]. Vaccines and therapies are important measures, despite some problems with social acceptance. Yet, they take time to develop and deploy widely and may wane in effectiveness as pathogens evolve, and thus their use holds little bearing on the importance of engineering controls for population protection. Yet all available control measures can help as part of a layered approach and may be required for extremely infectious agents.

As demonstrated by Nardell and colleagues in the case of TB [75], and Bueno de Mesquita and colleagues in the case of influenza [63], there exist potential limits to the extent that ventilation controls alone can control transmission risk in shared air environments. Given that seasonal influenza epidemics cause substantial burden of morbidity and mortality, and COVID-19 and other emerging pandemic pathogens can exact an even more devastating toll, there is a great opportunity for wider use of infection control via engineering controls with the greatest effectiveness. SARS-CoV-2 subvariants appear to be increasing in infectivity and a super spreading event suggests that a highly infectious case could produce over 1,000 quanta per hour [65]. This compares with measles cases which may shed 500 to over 5,000 quanta per hour [19, 64] and an influenza supershedder who generated approximately 600 quanta per hour [63]. Yet upper-room GUV has been shown to mitigate measles spread in elementary schools [76–78] and provide many times the equivalent sanitary ventilation provided by outdoor airflow and filtration [23, 79, 80]. Newer far-UVC applications that allow safe exposure to the light directly have been shown to have similar levels or better air disinfection with potential for widespread use in a greater variety of settings [81, 82]. The use of GUV technology offers to reduce the theoretical limits of sanitary ventilation and lowers transmission risk in congregate settings where filtration, outdoor airflow, and masking may not offer sufficient protection.

*Infectious Diseases Annual Volume 2022*

## **Author details**

Jacob Bueno de Mesquita Lawrence Berkeley National Laboratory, Berkeley, CA, USA

\*Address all correspondence to: jbuenodemesquita@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Airborne Transmission and Control of Influenza and Other Respiratory Pathogens DOI: http://dx.doi.org/10.5772/intechopen.106446*

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*Edited by Katarzyna Garbacz, Tomas Jarzembowski, Yuping Ran, Amidou Samie and Shailendra K. Saxena*

This book is an annual collection of reviewed and relevant research chapters authored by various researchers, offering a comprehensive overview of recent studies and developments in the field of Infectious Diseases. It joins work from four areas in this field, Bacterial, Fungal, Parasitic, and Viral Infectious Diseases, each edited by an expert. The global challenges and different living circumstances for people around the globe ask for a deep understanding of various aspects and properties of infectious diseases to ensure a safer and healthier cohabitation of humans and pathogens.

> *Alfonso J. Rodriguez-Morales, Infectious Diseases Series Editor*

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*Edited by Katarzyna Garbacz, Tomas Jarzembowski, Yuping Ran, Amidou Samie* 

*and Shailendra K. Saxena*