Morphometric and gross anatomy studies of gonads in day-old Arabic chick (Gallus turcicus) lead to gonadal asymmetry

The differentiation of avian gonads occurred since the embryonic stage, resulted in asymmetric morphology. This study aimed to analyse the morphometric and gross anatomy of gonads in Day old Arabic chick. This study utilized 116 Day old Arabic chick. After necropsy, they were divided into 61 male and 55 female. The variables studies were length, width, volume, location, colour, and shape of the gonads. Data analysis used descriptive analysis and independent T test. The results showed a significant difference (P<0.01) between the right and left ovary lengths 1.67±0.08 mm and 3.69±0.05, respectively. A significant difference (P<0.01) between the width ovaries 0.64±0.03 mm (right ovary) and 1.18±0.03 mm (left ovary), respectively. It was found a significant difference (P<0.05) between the length of right and left testis 2.47±0.08 mm and 3.29±0.07mm, and between volume of testis 1.46±0.12 mm3 (right testis) and 2.03±0.13 mm3 (left testis). However, the right and left testes width was not significant (P>0.01). The testes colour was creamy white, sometimes pink, and grey to black, the colour of ovaries was creamy yellow and pink. Morphometric and gonadal morphology of Day old Arabic chick showed gonadal asymmetry, and the left side is larger than the right side.

Introduction
Arabic chicken is one of the native chickens, layer-type chicken with superior egg production [1]. Its production potential has attracted farmers, so it has grown widely in several areas in Indonesia. In addition to having high egg production, Arabic chickens can adapt to various environmental conditions. Arabic chicken also does not have the brooding trait to extend their egg-laying period [2,3].
Freshly hatched Arabic chickens are generally monomorphic, difficult to determine between males and females because they have almost the same phenotypic characteristics. Recently, research has been developed about sexing Day-old-chick in Arabic chickens to distinguish sex quickly and accurately. Research on Day-old Arabic chick sexing requires proof of sex as soon as possible. One of the proofs is through gonad identification (postmortem) in day-old chicks because they are relatively more available than molecular sexing [4]. Gonads are reproductive organs, ovaries to produce ova in females, and testes to produce spermatozoa in males [5]. Because the size of the gonads of freshly hatched chickens is relatively small, it is necessary to observe their morphology to determine the testes or ovaries.
The avian gonads have location and morphology differences from mammals; birds' testes are in the abdominal cavity, normally, the right ovary regresses. The left ovaries have been identified as functional gonad, and asymmetric development of the right and left ovaries has been reported since the embryonic phase [6]. The male, both gonads develop into testes [7]. Testicular asymmetry is also common in poultry, especially in passerines, and the degree of asymmetry decreases with the increasing age of chickens. The right and left testes are functional, although the left is larger in most species [8]. The size of the testes is often associated with their reproductive ability [9]. Testicular morphometric studies have been carried out on adult Nigerian local chickens under physiological conditions to predict the reproductive ability of the testes [10]. Left-Right (L:R) asymmetry could be detected in both sexes in chickens [11].
Based on the description, it is necessary to reveal the shape and size of the ovary and testes of Day-old Arabic chick. This study aimed to analyse the morphometric and gross anatomy of the testes and ovaries of Day-old Arabic chick. Descriptions and information about their morphological characters are essential to confirm gonadal observations for purposes of proving to sex, to predict male quality through post-hatching development, and detect the gonadal abnormalities in Day-old chick (DOC).

Day-old-chick Arabic chicken
This study used 116 Day-old Arabic chick, resulting in mating between hen and Arabic gold rooster in previous studies [4]. Day-old chick obtained from the chicken breeding unit in Politeknik Pembangunan Pertanian Malang. The research was conducted at the Poultry Unit and the Reproduction Laboratory of the Politeknik Pembangunan Pertanian Malang.
The chicks have been estimated for their sex by feather sexing, then were euthanized by cervical dislocation. The necropsy procedures incise the ventral abdomen until the peritoneal cavity opens and removes the viscera organs, so the gonads are visible. The testes and ovaries were not removed from the abdominal cavity and were not weighed because relatively very small. The chicken was fixed in a dorsal lying position with the gonads still attached to the abdominal cavity, observed with direct eyes and then clarified under a trinocular stereo microscope connected to a computer to identify the gonads is easier.

Research variable
The gonads were examined for colour, shape, and location. Ovarian morphometrics includes the length and width of the right and left ovaries, while the testes include length, width, and volume. Data collection on the shape, colour, and location of the gonads was carried out under a stereo microscope with 10x magnification. The stereo microscope used is the Euromax SB 1903-P series produced by Holland with a DC 5000P camera. Measurement of the length and width of the gonads using the micrometre software from the camera. Quantitative data on testicular length and width were tabulated to calculate testicular volume.
Length of testes and ovaries was measured from the anterior end to the posterior end of the gonad, and gonad width was measured at the broadest part of the gonad. Testis's shape is ellipsoid [9], so the testicular volume was calculated using formula = (4/3) x π x (a) 2 (b). Where (a) is: half of the width and (b) is half of the length [12] or with formula (simplified) namely testicular volume = 0.523 x(Width) 2 (Length) [8].

Statistical analyses
The results of the gross anatomy identification of testes and ovaries were shown with explanation descriptions. The morphometric data were analysed using descriptive analysis. Differences in size between the right and left gonads were analysed by independent T-test at significance P<0.01, using SPSS version 16. Data was presented in mean ±SEM.

Gonads overview of day-old Arabic chick
Freshly hatched chicks were identified to be 55 females and 61 males after necropsy. The testes and ovaries were located in the abdominal cavity and could be seen when the viscera organs were removed. Results of observations to the ovaries and testes morphology are shown in Figure 1. Postmortem examination of chick testes and ovaries showed that the gonads consist of two right and left at above the surface of the kidney. The gonads were connected to the backbone on the surface of the two kidneys.

Morphometric and Gross Anatomy of Day-old Arabic Chick Ovaries
Gross anatomy observation of the gonads showed that the shape of the ovary is irregular, and the left ovary has a more defined shape than the right ovary. The ovaries are close to the adrenal glands, where the right side is caudolateral of the adrenal gland, while the left side covers the ventral adrenal gland. The ovaries are located in the ventromedial mesonephros although the mesonephros is sometimes not visible due to regression.
In general, the ovaries of Day-old Arabic chick have an elongated shape with slightly curved laterally or towards the kidneys. The left ovary shows a curvature towards the right kidney with various shapes and uneven edges. The right ovary is smaller with an elongated and irregular shape. Almost all of the left adrenal glands are covered by the ovaries, and the right adrenal gland is more visible. The colour of the ovaries is creamy yellow, transparent pink, and sometimes grey. The surface of the left ovary appears smoother, slightly compact. This ovarian character is still similar to the morphology of the ovaries of 1-week old chickens, as described by [6]. The gross anatomy of the ovary of Day-old Arabic chick was shown in Figure 2.  , part (a) the shape of the right ovary: small elongated, the right mesonephros appears small, and the left mesonephros is not visible; (b). the left ovary is much larger, the right and left mesonephros are still visible clearly; (c) the right ovary is like a bean arch, the right, and left mesonephros are still visual even though they are smaller; (d). The right ovary is small, like a bean arch and the adrenal gland and the right mesonephros are not visible. The difference between the right and left ovaries: the left ovary is more elongated than the right ovary, anterior end of the left ovary is wider than the posterior end. The left ovary appears thinner than the right ovary. The T-test between right and left ovary length P-value <0.0001** The T-test between the width of the right and left ovaries P-value <0.0001** Note: **: indicates a significant difference (P<0.01) Table 1 shows that the length and width of the ovaries vary widely. T-test analysis showed that the size difference between the right and left ovaries was very significant (P<0.01). The size difference of the chick ovary results from the asymmetry process that has occurred since the embryonic phase. In domestic birds, the left and right ovaries begin to grow around 72 hours of initial incubation [13] and morphological differentiation of gonads occurred on day 6.5 of incubation [5]. In the early stages before sexual differentiation, the ovaries of chicken embryos develop symmetrically without showing significant left-right asymmetry. As the embryo develops, the right ovary in the medulla develops without a cortex. This cortical development is considered essential for developing the left ovary [13]. There is a size difference between the right and left ovaries, resulting in ovarian asymmetry on the 20th day of incubation [6] [14]. From this study, ovary asymmetry in Arabic chicken was identified after hatching. The hen has only one ovary and oviduct on the left side [5].

Morphometric and Gross Anatomy of Day-old Arabic chick testes
Gross anatomy observations showed the testes are located in the ventromedial mesonephros. The mesonephros has regressed from the embryonic phase at hatching. It appears as a small part at the bottom of the gonads. The invasion of primordial germ cells forms embryonic gonads into the coelomic epithelium that partially covers the mesonephros [15]. The cranial portion of the testes is located close to the ventral border of the lungs. In contrast, the caudal part is located in the cranial-ventral kidney.
The colour of the testes in Arabic chicken was creamy white, slightly pink, grey, and some were black in the left or right testicles, or even black in both right and left testes. The testicles of certain breeds of fowl may be mixed with black colour and white patches or maybe entirely greyish black due to the presence of the pigment melanin in the melanoblasts, in the connective tissue of the testes [14]. The gonads of sexually immature or resting birds (in seasonal birds) have yellowish-white to black or grey colouration, becoming moderately black in regressing testes and containing melanoblasts [14]. Testicular colour variability can be caused by sexual activity in testes and chicken's age. Bright yellow in juveniles and grey-white in adults of the quails and ostrich. The bright yellow colour in juveniles is due to the accumulation of lipids in the interstitial cells and the grey-white colour in adults due to the spread of interstitial cells by enlarging seminiferous tubules after sexual maturity [20]. The results of this study, most of the testicle's colour in DOC Arab chickens is yellow-white (creamy), the findings of the other colours besides bright yellow, still have to be confirmed histologically to support the determination of its quality.
The description of the location, shape, and colour of the Day-old Arabic chick testes is presented in Figure 3. The testes of birds in this study are bean-shaped and elongated ovals, generally belonging to the category of an ellipsoid or prolate spheroid shape [8]. The form of the testes tends to be straight, lengthwise cranial and caudal, or slightly curved towards the kidney. Figure 3 shows that (a) the left testis is larger than the right testis, the right and left sides show clear mesonephros; (b) the size of the right testis is larger than the left testis; (c). The length of the right testis is almost the same as that of the left. However, the left testis is bigger and grey with a wider anterior end and black colour (d). the left testis is longer than the right testis, most of the left testis is darker in colour, and the anterior end of the right testis is grey. The results on Day-old Arabic chick, the number of detected black colour on the testes was 22.95%. Melanoblasts cause dark colour in the interstitium, so the testes are melanistic. Bicoloured testes are not considered gonads abnormal. The testes with bicolour do not show any histologic abnormalities in African flamingos [16]. In this study, the position of the right and left testes tends to be symmetrical, but there are differences in size. The morphometrics of Day-old Arabic chick is presented in Table 2. Table 2 showed the size (length, width and volume) of left testis is larger than right testis. The results of the independent T-test analysis showed that the length and volume of the testes were significantly different between the right and left testes (P<0.01). In contrast, the testes' width was not significantly different between the right and left (P>0.01). This study showed that the number of Day-old Arabic chick with the left testis was larger than the right, namely 72.13%, and the number of Day-old Arabic chick with the right testis was larger than the left testis only 27.86%. It has been described in previous study that the left testis larger than the right in birds commonly, but there are exceptions in some species where there is no difference in size, and the right testicle is larger than the left testis [14]. The left testicle is larger than the right testis in Gallus domesticus is 63% in birds and 57%.
The right testis is larger than the left testis, only 26.5%, while both testes are the same in 8.2% of birds [8]. Testicular asymmetry may be caused by irregular migration of the Primordial Germ Cells (PGC) to the left and right sides during embryonic development. This testicular asymmetry has been reported that the right gonadal cortex loses its chemotactic attraction to PGCs. Thus, it leads to the left gonadal cortex on day 3th of embryogenesis [14]. Similar results were reported by [11] and [18] that the number of PGCs in the left testis is more than in the right testis, and in the chicken embryos on the 6th day of incubation, the size of the left testis relatively larger. Despite the lack of a relationship between male quality and the degree of testicular asymmetry, but several studies have found that testicular asymmetry increases with age [19]. The results of this study can be used as a basis for Day-old chick testicular morphometrics to be compared with the age afterward in the same type of chicken.

Conclusion
Studies of Gonad morphometrics in Day-old Arabic chick have shown gonadal asymmetry, left ovary are larger than the right ovary. The size and volume of the left testis are larger than the right testis. Gross anatomy and evidence of gonadal asymmetric in Arabic chicken can be used as reference when observing the gonads and as information on the gonad's development at hatching. Further research is needed to reveal the histological and molecular asymmetry, and the observation of gonadal size at a later age, which can be associated with the quality of the chicken.


s2fieldsofstudy: Biology Medicine

text:
Humoral and Cellular Responses to COVID-19 Vaccines in SARS-CoV-2 Infection-Naïve and -Recovered Korean Individuals

In the face of a global COVID-19 vaccine shortage, an efficient vaccination strategy is required. Therefore, the immunogenicity of single or double COVID-19 vaccination doses (ChAdOX1, BNT162b2, or mRNA-1273) of SARS-CoV-2-recovered individuals was compared to that of unvaccinated individuals with SARS-CoV-2 infection at least one year post-convalescence. Moreover, the immunogenicity of SARS-CoV-2-naïve individuals vaccinated with a complete schedule of Ad26.CoV2.S, ChAdOX1, BNT162b2, mRNA-1273, or ChAdOX1/BNT162b2 vaccines was evaluated. Anti-SARS-CoV-2 S1 IgG antibody (S1-IgG), pseudotyped virus-neutralizing antibody titer (pVNT50), and IFN-γ ELISpot counts were measured. Humoral immune responses were significantly higher in vaccinated than in unvaccinated recovered individuals, with a 43-fold increase in the mean pVNT50 values. However, there was no significant difference in the pVNT50 and IFN-γ ELISpot values between the single- and double-dose regimens. In SARS-CoV-2-naïve individuals, antibody responses varied according to the vaccine type: BNT162b2 and mRNA-1273 induced similar levels of S1-IgG to those observed in vaccinated, convalescent individuals; in contrast, pVNT50 was much lower in SARS-CoV-2-naïve vaccinees than in vaccinated recovered individuals. Therefore, a single dose of ChAdOX1, BNT162b2, or mRNA-1273 vaccines would be a good alternative for recovered individuals instead of a double-dose regimen.

Introduction
Since the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was reported in December 2019, there have been more than 318 million confirmed cases of coronavirus disease (COVID-19) as of January 2022, including 5.5 million deaths worldwide [1]. The COVID-19 pandemic accelerated the development and production of several vaccine platforms, such as mRNA, viral vector, inactivated, subunit, and DNA vaccines. Until now, the WHO has approved ten COVID-19 vaccines for emergency use, i.e., two mRNA vaccines-BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna)three non-replicating viral vector vaccines-ChAdOX1 (Oxford/AstraZeneca), Covishield (Oxford/AstraZeneca/Serum Institute of India), and Ad26.COV2.S (Janssen)-three inactivated vaccines-Covaxin (Bharat Biotech), CoronaVac (Sinovac), and BBIBP-CorV (Sinopharm)-and two protein subunit vaccines-NVX-CoV2373 (Novavax) and COVO-VAX (Novavax/Serum Institute of India) [2]. Most countries implemented mass vaccination campaigns to achieve herd immunity and reduce morbidity and mortality. With the fourth COVID-19 wave progressing worldwide and the increasing threat of new variants, extensive vaccination programs should be implemented to maximize efficient vaccine provision with limited vaccine resources. Most recovered patients already have some immunity against SARS-CoV-2, although it is insufficient to protect against variants. The currently available COVID-19 vaccines designed against the spike protein of wild-type SARS-CoV-2 effectively protect against severe COVID-19 and death caused by the variants [3][4][5][6]. However, the imbalance between the demand and supply of COVID-19 vaccines is a serious concern, especially in low-income countries. Additional doses have been administered to individuals who have received a complete vaccine dose in high-income countries. Only 13.6% of the population had received at least a single vaccine dose in Africa as of 5 January 2022 [7]. Therefore, strategies for effective vaccine provision should be considered.
In order to evaluate the humoral and cellular immune responses elicited by ChAdOX1, BNT162b2, and mRNA-1273 vaccines in recovered individuals, we performed age-, sex-, and initial symptom onset (ISO)-matched case-control studies between a single-dose-vaccinated group and an unvaccinated group of recovered individuals. Moreover, we compared the immunogenicity of mRNA and viral vector vaccines in SARS-CoV-2-naïve individuals.

Participants and Study Design
To evaluate vaccine-induced immunity in recovered and infection-naïve healthy individuals, we selected 52 participants from the YUMC-COVID-R02 study (IRB No. YUMC-2020-04-009), an ongoing longitudinal immunogenicity evaluation study of COVID-19convalescent individuals, and 62 healthy SARS-CoV-2-naïve individuals were newly enrolled (IRB No. YUMC-2021-03-012). COVID-19-convalescent patients had been diagnosed using RT-PCR during the first wave of COVID-19 (from February 2020 to April 2020) [19]. They had been discharged from either hospitals or residential treatment centers and visited Yeungnam University Medical Center four times for blood sampling after recovery. Among them, 18 were vaccinated with one dose, and 17 were vaccinated with two doses of COVID-19 vaccines (ChAdOX1, BNT162b2, mRNA-1273, or ChAdOX1/BNT162b2 vaccines) between visits 3 and 4, at least one year after recovery, except for one individual. Convalescent individuals with a single dose or without vaccination were matched for age, sex, and number of days after ISO at time of blood collection and nested case-control analyses were performed. Blood samples were collected from SARS-CoV-2-naïve individuals within 1 week before vaccination and 4-5 weeks after the completion of the vaccination schedule. Plasma was stored at −80 • C, and peripheral blood mononuclear cells (PBMCs) were cryopreserved in liquid nitrogen until use.

Anti-SARS-CoV-2 IgG Enzyme-Linked Immunosorbent Assay (ELISA)
To detect anti-SARS-CoV-2 S1 and nucleocapsid protein (NCP) IgG antibodies, plasma samples were tested with EUROIMMUN ELISA SARS-CoV-2 IgG (EUROIMMUN AG, Lübeck, Germany) coated with recombinant SARS-CoV-2 spike protein (S1 domain) or NCP. ELISA was performed according to the manufacturer's instructions. Optical density (OD) was measured at a wavelength of 450 nm with a reference wavelength of 620 nm using a microplate reader (Multiskan FC, Thermo Scientific, Waltham, MA, USA). The results were evaluated semiquantitatively by calculating the ratio between the extinction of the control or sample and that of the calibrator. The ratio was calculated using the following formula: Ratio = Extinction of the control or sample/extinction of calibrate Samples with a ratio of <0.8 were interpreted as negative, 0.8-<1.1 were borderline, and ≥1.1 were positive. Then, 50% of the SARS-CoV-2 pseudovirus-neutralizing antibody titer (pVNT 50 ) was determined by non-linear regression using GraphPad Prism 9.3.0 (GraphPad Software, Inc., San Diego, CA, USA). pVNT 50 below the detection limit was recorded as 10 and was considered the cutoff titer.

Enzyme-Linked Immunospot (ELISpot) Assay
The cryopreserved PBMCs were thawed, and 2.5 × 10 5 PBMCs/well in a 96-well plate were stimulated with 0.16 µg/mL SARS-CoV-2 M-, N-, and S-protein overlapping peptide pools (Miltenyi Biotec, San Diego, CA, USA) for 18 h at 37 • C in an atmosphere of 5% CO 2 ; cells stimulated with anti-CD3 mAb (100 ng/mL; MABTECH AB, Stockholm, Sweden) were used as positive controls; cells stimulated with the medium (RPMI 1640, Lonza) alone were used as negative controls. IFN-γ spot-forming counts (SFCs) were detected using a human ELISpot PRO kit (MABTECH) according to the manufacturer's instructions. The number of spots was counted using an ImmunoSpot Counter (CTL-ImmunoSpot, Cleveland, OH, USA).

Statistical Analysis
Data were analyzed and plotted using GraphPad Prism 9.3.0 (GraphPad Software, Inc., San Diego, CA, USA). All datasets were tested for statistical normality, and this criterion was used to decide the appropriate (parametric or nonparametric) statistical test. The Mann-Whitney test and Wilcoxon matched-pairs signed-rank test were used to compare the differences between the experimental groups. Multiple comparisons were performed using two-way ANOVA, one-way ANOVA, and Kruskal-Wallis tests for statistical analysis (significance levels: not significant (ns): p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Immune Responses in SARS-CoV-2-Recovered Individuals
To evaluate vaccine-induced immunity in SARS-CoV-2-recovered individuals, 35 individuals were selected for a nested case-control study fromYUMC-COVID-19-R02, a longitudinal immunogenicity follow-up study of COVID-19-recovered patients ( Table 1). Eighteen individuals were vaccinated with a single dose before their fourth visit (mean = 497.9 days after ISO) (eleven with ChAdOX1, four with BNT162b2, and three with mRNA-1273). The mean number of days after vaccination at the time of blood sampling was 30.7 days (range: 9-66). Seventeen age-, sex-, and days after ISO-matched unvaccinated SARS-CoV-2recovered individuals were selected from the YUMC-COVID-19-R02 study. The mean age of the vaccinated COVID-19-recovered individuals was 56.4 years (range: 40-71), while that of the unvaccinated individuals was 53.3 years (range: 34-70). Sixteen out of the eighteen vaccinees and fifteen out of the seventeen unvaccinated individuals were women. There were 14 and 2 cases of mild and moderate disease symptoms, respectively, and one each of asymptomatic and severe disease in vaccinated individuals based on the National Early Warning Score (NEWS). All the unvaccinated individuals had mild disease symptoms. The longitudinal study of the natural immunity against COVID-19 showed that anti-SARS-CoV-2 S1 binding antibody titers (S1-IgG) gradually decreased in most individuals from visit one (mean = 143.9 and 146.6 days after ISO in unvaccinated and single-dosevaccinated groups, respectively) to visit three (mean = 331.9 and 358.7 days after ISO in unvaccinated and single-dose-vaccinated groups, respectively) (Table 1, Figure 1, left column). The mean S1-IgG was not significantly different between the unvaccinated and vaccinated groups prior to vaccination (5.2 ± 1.8 vs. 4.6 ± 2.5, 3.6 ± 1.5 vs. 3.5 ± 1.8, and 3.3 ± 1.6 vs. 3.0 ± 1.5 at visits one, two, and three, respectively; p > 0.05, Figure 1a, Table 2). However, the level of S1-IgG was significantly increased after a single vaccination dose (compared to the unvaccinated groups; 2.8 ± 1.6 vs. 11.2 ± 1.0; p < 0.0001, Figure 1a, Table 2). The mean pVNT 50 was 131 ± 124 vs. 97 ± 133, 100 ± 98 vs. 52 ± 46, 86 ± 84 vs. 106 ± 235, and 77 ± 95 vs. 3333 ± 2322 at visits one, two, three, and four, respectively, in the unvaccinated and single-dose-vaccinated groups (Figure 1b, Table 2). The neutralizing antibody titer was also not significantly different from visits one to three between the unvaccinated and single-dose-vaccinated groups ( Figure 1b); however, it was significantly increased after a single-dose vaccination (visit four) in the recovered individuals (p < 0.0001). The cellular immunity measured based on the levels of IFN-γ SFCs was not different between the unvaccinated and single-dose-vaccinated SARS-CoV-2-recovered individuals during all the visits (39.9 ± 49.5 vs. 87.9 ± 188.1, 26.7 ± 21.0 vs. 57.8 ± 101.0, 27.2 ± 42.6 vs. 31.6 ± 38.6, and 23.8 ± 27.6 vs. 46.0 ± 44.3 at visits one, two, three, and four, respectively; p > 0.05, Figure 1 right column, Table 2). Therefore, vaccination boosted humoral immunity, represented by the increase in the levels of binding (S1-IgG) and neutralizing antibodies (pVNT 50 ), by 4-and 43-fold, respectively. At the same time, the counts of IFN-γ SFCs were only 1.9-fold higher in the vaccinated group than in the unvaccinated group of SARS-CoV-2-recovered individuals ( Table 2). Next, we compared the differences in humoral and cellular immunity between the single-(n = 18) and double-dose-(n = 17) vaccinated SARS-CoV-2-recovered individuals ( Figure 2, Table 3). The mean age of the double-dose-vaccinated COVID-19-recovered individuals was 56.7 years (range: 23-79); among these, 12 individuals were women. Among the 17 individuals who received double-dose vaccines, 14 had mild disease, 2 were asymptomatic, and 1 had moderate disease symptoms. The mean number of days after ISO during the second dose administration was 538.3 days (range: 430-642 days). The mean number of days after the second-dose vaccination at the time of blood sampling was 72.7 days (range: 7-141). The mean S1-IgG, pVNT 50, and IFN-γ SFCs were 11.2 ± 1.0 vs. 12.4 ± 1.4, 3333 ± 2322 vs. 2914 ± 2591, and 46.0 ± 44.3 vs. 26.1 ± 32.8 in singleand double-dose vaccinees, respectively. Even though the binding antibody titers differed between single and double doses, the neutralizing antibody and cellular immunity were not significantly different between these regimens in SARS-CoV-2-recovered individuals (p > 0.05) (Figure 2, Table 3).

Immune Responses in SARS-CoV-2-Naïve Individuals
To evaluate vaccine-induced immunity in SARS-CoV-2-naïve individuals, 62 individuals who neither had been diagnosed with COVID-19 nor had any related symptoms were enrolled. Fifty-six individuals were vaccinated with a complete schedule of Ad26.CoV.S, ChAdOX1, BNT162b2, or mRNA-1273, and six individuals were crossvaccinated with primary ChAdOX1 and secondary BNT162b2 doses (Table 4). According to the Korean government's COVID-19 vaccine policy, Ad26.CoV2.S was administered to young men, and ChAdOX1 was mainly administered to individuals older than 50 years of age. Therefore, the age and sex distributions were not even in the five vaccine groups; nevertheless, the total sex ratio was similar, i.e., 30:32 in women and men (Table 4). CoV-2 S1 IgG ELISA ratio; (h) pVNT 50 titer; (i) IFN-γ ELISpot counts from visit one to visit four in single-dose-vaccinated individuals from the longitudinal study. The blue dashed line indicates the cutoff (S1-IgG = 0.8 for negative and 1.1 for positive, pVNT 50 = 10). The significance of the differences between the unvaccinated and single-dose-vaccinated groups was determined using two-way ANOVA by Holm-Šídák's multiple comparisons test. ns, p > 0.05, **** p < 0.0001.

Discussion
Here, we performed an age-, sex-, and ISO-matched nested case-control study between a single-dose-vaccinated group and an unvaccinated group of SARS-CoV-2-recovered individuals, in addition to a comparison with infection-naïve individuals. Case-control groups showed similar S1-IgG, pVNT 50 , and IFN-γ spot counts before vaccination (from visits one to three). However, the humoral immune response, especially the neutralizing antibody, was significantly increased after a single-dose vaccination compared to the unvaccinated, recovered group. Therefore, protective immunity seems to be significantly strengthened by COVID-19 vaccination in SARS-CoV-2-recovered individuals. A single dose of BNT162b2, mRNA-1273, or Ad26.COV2.S induced higher anti-S antibody titers in the recovered individuals than in the unvaccinated convalescent individuals, regardless of the vaccine type [20]. In our study comprising 18 individuals, 61% of the single-dose vaccinees were administered ChAdOX1, and the others were administered mRNA vaccines: 22% BNT162b2 and 17% mRNA-1273. Similar to the conditions observed in response to Ad26.COV2.S [20], no significant immunological differences were observed between the ChAdOX1 and mRNA vaccines, regardless of the dose, in the recovered individuals ( Figures S1 and 2). Ebinger et al. reported that the anti-S RBD antibody titer and ACE2binding capacity after a single dose were similar to the response seen after two doses of the BNT162b2 vaccine in previously infected healthcare workers [9]. In addition, Krammer et al. also reported no difference in anti-spike antibody response after the first and second dose of mRNA vaccine in COVID-19-recovered individuals [10]. Therefore, a single-dose regimen of either ChAdOX1 or mRNA vaccines might be suitable for these individuals.
In COVID-19-naïve individuals, the humoral immune response vastly differed according to the vaccine type. Ad26.CoV2.S induced the lowest, and mRNA-1273 induced the highest immune responses compared to the other vaccines ( Figure 3). In particular, the seroconversion rate of the neutralizing antibodies was very low (20%) in the Ad26.CoV2.S vaccinees, but it was 84.6% and 100% in the ChAdOX1 vaccinees and all the other types of vaccinees, respectively (Figure 3b). These data were consistent with the previous findings that the individuals who received the complete mRNA vaccine dose were all seropositive in the SARS-CoV-2 pseudovirus neutralization test. In contrast, the seropositive rate of Ad26.CoV2.S vaccines was only 9.5% [20]. According to previous data, the seroprevalence rate of neutralizing antibodies against human adenovirus type 26 (AdHu26) showed distinct geographic distributions, such as 35.5% (407/1154) in China [21], 64.6% in South Africa, and 11.7% in the USA [22]. Unfortunately, there were no seroprevalence data for AdHu26 in Korea. We could not explain whether the preexisting AdHu26 immunity influenced the immunogenicity of Ad26.CoV2.S. Despite the low prevalence of the AdHu26 antibody, the aforementioned AdHu26 seroprevalence data did not represent seroprevalence in the entire USA; the seropositive rate of Ad26.CoV2.S in the USA was also very low. Interestingly, ChAdOX1 induced a broad range of humoral immune responses compared to either mRNA or ChAdOX1/BNT162b2 vaccines in COVID-19-naïve individuals (Figure 3a,b); a similar phenomenon was reported by Jeewandara et al. [17]. Nevertheless, ChAdOX1 induced uniform humoral immune responses in the recovered individuals, which were similar to those observed in response to mRNA-1273 or BNT162b2. Therefore, ChAdOX1 might be more effective in COVID-19-recovered individuals than in infection-naïve individuals.
We analyzed the data from pre-and post-vaccination with ChAdOX1, BNT162b2, or mRNA-1273 vaccines and visit four in the recovered individuals. The anti-S1 antibody, pVNT 50 , and IFN-γ ELISpot counts differed significantly between the unvaccinated infection-naïve group and the previously unvaccinated, infection-naïve vaccinated, and recovered vaccinated groups ( Figure S2, Table S2). Therefore, the three vaccines induced significantly different immunogenicity in infection-naïve and recovered individuals. However, the small number of vaccinees is a limitation of this study.

Conclusions
A single dose of ChAdOX1, BNT162b2, or mRNA-1273 COVID-19 vaccines induced significantly high levels of anti-S1 and neutralizing antibodies in SARS-CoV-2-recovered individuals. No immunological differences were observed between single-and doubledose vaccine regimens in such cases. However, Ad26.CoV2.S and ChAdOX1 induced much lower anti-S1 and neutralizing antibody levels than BNT162b2 or mRNA-1273 vaccines in SARS-CoV-2-naïve individuals. Because ChAdOX1 has several benefits, such as low cost and stability of refrigerating temperature, it would be beneficial to administer the ChAdOX1 vaccine to the SARS-CoV-2-recovered individuals in tropical areas or low-income countries. BNT162b2 and mRNA-1273 vaccines induced significant cellular immunity in SARS-CoV-2-naïve individuals (compared to pre-vaccination), but not in the recovered individuals, even though the mean IFN-γ spot counts were increased after vaccination. These data demonstrate significant variations in the immunogenicity of the currently used COVID-19 vaccines in Korea and suggest a different vaccination strategy in infection-naïve and recovered individuals.


Synergistic Anti-tumor Effect of Dichloroacetate and Ivermectin

We formerly reported that the combination of dichloroacetate, omeprazole, and tamoxifen blocked cancer progression by reducing lactic acid production and inducing superoxide production. Recently, ivermectin, a well-known anti-parasite drug, was reported to share the same mechanisms with them and have anti-tumor activity. Here, we present three patients in whom the combination of dichloroacetate, omeprazole (plus tamoxifen), and ivermectin dramatically relieved the symptoms accompanying cancer and sarcoma progression.

Introduction
An increasing number of clinically approved non-cancer drugs have been proved to have antitumor activity. Among those was dichloroacetate, an approved drug for congenital lactic acidosis [1]. Warburg discovered that even in the presence of sufficient oxygen, cancer cells metabolize glucose and produce lactic acid [2][3][4]. Bonnet et al. reported that dichloroacetate reverses the Warburg effect and inhibits tumor growth [5]. Dichloroacetate increases the flux of pyruvate into the mitochondria by inhibiting pyruvate dehydrogenase kinase and promotes glucose oxidation over glycolysis. As a result, dichloroacetate decreases the production of lactic acid and increases superoxide production, which relieves the symptoms accompanying cancer progression and induces mitochondria-dependent apoptosis.
We formerly reported that omeprazole (a proton pump inhibitor) enhances the antitumor effect of dichloroacetate by the alkalinization of lysosomes and the permeabilization of lysosome membranes followed by the production of reactive oxygen species [6][7]. Tamoxifen also induces caspase-dependent cancer cell growth inhibition through superoxide production and is used not only for hormone receptorpositive breast cancer but also for pancreatic cancer and sarcoma [8][9]. Taken together, we showed that the addition of tamoxifen to the combination of dichloroacetate and omeprazole strengthens their effects on fibrosarcoma cells in vitro [10]. This combination also blocked cholangiocarcinoma progression for three months [10].
On the other hand, ivermectin, an avermectin derivative used to treat parasites, was reported to have a growth inhibitory effect on cancer cells through the induction of mitochondrial dysfunction and oxidative damage [11][12]. Since ivermectin shares the same anti-tumor mechanism as dichloroacetate, omeprazole, and tamoxifen, we postulated that a full or partial combination of these drugs might be a promising protocol for treating malignant tumors.

Case Presentation
Here, we show three cases successfully treated with a full or partial combination of dichloroacetate, omeprazole (plus tamoxifen), and ivermectin.

Case 1
A 69-year-old female was diagnosed with invasive breast cancer in October 2015. She underwent left breast partial mastectomy, but bone metastases and pleural dissemination appeared ( Figure 1). She responded to chemo-endocrine therapy, but it soon became less effective, and intolerable side effects appeared. We gave up conventional therapy and started treatment with dichloroacetate, omeprazole, and tamoxifen from October 2021: weekly use of three tablets of 333 mg dichloroacetate per day (on Day 1), three tablets of 40 mg omeprazole per day (on Day 1), along with a tablet of 20 mg tamoxifen every day. This relieved her symptoms (bone pain, shortness of breath, and general fatigue) instantly but not completely. Hence, we added a tablet of 12 mg ivermectin per day (on Day 1), which stabilized pleural effusion and induced tumor marker reduction (CEA or carcinoembryonic antigen), which went down from 12.9 to 7.3, and cancer antigen 15-3 (CA15-3), which went down from 302.3 to 229.4 in three months).

FIGURE 1: CXR showing pleural effusion due to breast cancer dissemination
CXR: chest X-ray

Case 2
A 54-year-old male was diagnosed with right femur osteosarcoma in February 2021. He underwent neoadjuvant chemotherapy with adriamycin and ifosfamide followed by tumor resection but local recurrence occurred. Although he received chemotherapy with ifosfamide, doxorubicin, and cisplatin, lung lymph node metastases and pleural dissemination appeared ( Figure 2). He could not walk out of his house himself because of shortness of breath and severe pain. Since the combination of dichloroacetate, omeprazole, and tamoxifen had not been effective in sarcoma patients in our study, we decided to add ivermectin to them: weekly use of three tables of 333 mg dichloroacetate per day (on Days 1 and 4), three tablets of 40 mg omeprazole tablets per day (on Days 1 and 4), a tablet of 20 mg tamoxifen per day (on Days 1 and 4), along with a tablet of 12 mg ivermectin per day (on Days 1 and 4). After only one cycle, all the symptoms were relieved dramatically, and he could come to our clinic on foot by himself.

Case 3
A 54-year-old male was diagnosed with right lung adenocarcinoma in September 2014 ( Figure 3) and underwent right lung lobectomy. Although he underwent postoperative chemotherapy, local recurrence with lymph node metastases appeared in one year. Initially, chemotherapy was effective, but gradually, it became less effective and metastases in other sites (bone and brain) appeared. Even after we started the combination therapy with a tablet of 333 mg dichloroacetate every two days, a tablet of 40 mg omeprazole every two days, and a tablet of 12 mg ivermectin per week, his symptoms (cough, shortness of breath, pain, and appetite loss) did not improve. However, soon after we changed our protocol (a tablet of 12 mg ivermectin twice a week), all the symptoms were relieved.

Discussion
Cancer-associated pain is a major cause of increased morbidity and diminishes the quality of life. Cancer cells release protons and lactate via plasma membrane pH regulators to avoid the intracellular acidification resulting from increased aerobic glycolysis, known as the Warburg effect [2][3]. Since acidosis is algogenic for sensory neurons, the acidic microenvironments can evoke cancer-associated pain.
Dichloroacetate inhibits the Warburg effect. It inhibits pyruvate dehydrogenase kinase, which blocks pyruvate dehydrogenase through its phosphorylation activity [5]. When pyruvate dehydrogenase kinase is inhibited by dichloroacetate, pyruvate dehydrogenase is reactivated, causing the mitochondria to no longer be hyperpolarized; instead, the membrane and mitochondria are depolarized, reactivating the mitochondrial K+ channels, which then decrease cytosolic K+. When pyruvate dehydrogenase is inhibited in cancer cells by pyruvate dehydrogenase kinase, an excess cytosolic K+ occurs that inactivates caspases 3 and 9. Dichloroacetate reactivates these caspases along with an increase in hydrogen peroxide intracellularly, allowing the release of cytochrome c from the mitochondria. The release of cytochrome c is a major activating step for cell apoptosis, as it triggers the caspase cascade.
On the other hand, the postulated mechanisms of the antitumor effects of ivermectin are: 1) inhibition of the multidrug resistance protein P-glycoprotein, 2) osmotic cancer cell death by the upregulation of the chloride channel, 3) P2X4/P2X7-and caspase-1-mediated immunogenic cancer cell death, 4) inhibition of the PAK1/Akt/mTOR pathway, 5) inhibition of the WNT-TCF pathway, 6) blockade of the PAH2-SID interaction, 7) inhibition of the helicase activity, 8) reduction of cancer stem cell populations accompanying the downregulation of Nanog, Oct4, and Sox2, 9) induction of anti-angiogenic effects, and 10) mitochondrial dysfunction with oxidative damage [13].
Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in controlling cellular pH in normal and tumor cells. Treatment with proton pump inhibitors induces the sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. This effect of proton pump inhibitors is mediated by the very early production of reactive oxygen species. Tamoxifen also induces reactive oxygen species-mediated autophagic cell death [10].
Dichloroacetate, omeprazole, tamoxifen, and ivermectin share the same anti-tumor mechanism of mitochondrial dysfunction followed by reactive superoxide production. The full or partial combination of them was expected to reduce the necessary dose of each drug and the accompanying risks. Although dichloroacetate alone was reported to reduce severe cancer pain and rarely eradicate tumors [14][15], most chemotherapy-resistant cancer and sarcoma have not responded to the combination of dichloroacetate, omeprazole, and tamoxifen in our study [10]. However, in this report, we presented three cases in which the addition of ivermectin to the combination of dichloroacetate and omeprazole (plus tamoxifen) was effective. Although our rapid communication is preliminary, we think our protocol may open new therapeutic options.

Conclusions
We formerly reported that omeprazole and tamoxifen enhanced the antitumor effect of dichloroacetate by the production of reactive oxygen species, and this combination blocked cholangiocarcinoma progression for three months. Ivermectin, an avermectin derivative used to treat parasites, was reported to have a growth inhibitory effect on cancer cells through the induction of mitochondrial dysfunction and oxidative damage. Since all these drugs share the same anti-tumor mechanism, we postulated that a full or partial combination of these drugs might be a promising protocol, and in this report, we showed that combination therapy with dichloroacetate, omeprazole (plus tamoxifen), and ivermectin was extremely effective to relieve the symptoms accompanying cancer and sarcoma.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.


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Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection

The palatine tonsils (hereinafter referred to as “tonsils”) serve as a reservoir for viral infections and play roles in the immune system's first line of defense. The aims of this study were to establish tonsil epithelial cell–derived organoids and examine their feasibility as an ex vivo model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The tonsil organoids successfully recapitulated the key characteristics of the tonsil epithelium, including cellular composition, histologic properties, and biomarker distribution. Notably, the basal layer cells of the organoids express molecules essential for SARS-CoV-2 entry, such as angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin, being susceptible to the viral infection. Changes in the gene expression profile in tonsil organoids revealed that 395 genes associated with oncostatin M signaling and lipid metabolism were highly upregulated within 72 h after SARS-CoV-2 infection. Notably, remdesivir suppressed the viral RNA copy number in organoid culture supernatants and intracellular viral protein levels in a dose-dependent manner. Here, we suggest that tonsil epithelial organoids could provide a preclinical and translational research platform for investigating SARS-CoV-2 infectivity and transmissibility or for evaluating antiviral candidates.

Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, was first identified on December 2019 in Wuhan, China [1,2]. The emerging virus spread rapidly around the world, and the World Health Organization (WHO) declared the outbreak as a pandemic in March 2020. SARS-CoV-2 belongs to genus Betacoronavirus, which also includes the highly pathogenic Middle East respiratory syndrome (MERS)-and SARS-CoVs [3]. Infection with SARS-CoV-2, which is mainly transmitted through the upper respiratory tract, causes diverse disease symptoms ranging from mild sickness (including fever, cough, sore throat, and loss of olfaction or taste) to severe illness (including pneumonia and even death) [1,4,5]. Currently, mRNA-and adenoviral vector-based vaccines have been approved for prevention of SARS-CoV-2 infection, and antivirals such as remdesivir (polymerase inhibitor) and PF-07321332 (protease inhibitor) have been developed for treatment of infected patients [6][7][8][9][10]. However, viral variants with higher transmissibility or pathogenicity have been isolated with increasing frequency worldwide. Regarding the emerging and re-emerging rate of mutant CoVs, a suitable ex vivo SARS-CoV-2 infection model system is indispensable for development of broad-spectrum antivirals as well as universal vaccines or for understanding SARS-CoV-2 pathogenesis in a personalized manner [11][12][13][14].
Several reports have described the recapitulation of human organoids for SARS-CoV-2 infection and demonstrated their feasibility for analyses of antiviral activity or tissue susceptibility. For instance, human pluripotent stem cell (hPSC)-derived organoids have proven valuable for investigation of SARS-CoV-2 tropism [15,16]. In addition, SARS-CoV-2 infection has been evaluated in human intestinal, liver ductal, lung, and brain organoids [17][18][19][20][21]. However, most of these approaches use synchronized stem cells or human tissues obtained through conventional open surgeries, imposing limitations on clinical sample availability and research in personalized medicine. To address these issues, we attempted to establish an alternative organoid system for which i) human tissues could be reliably accessible with reduced physical burdens, ii) organoids can be maintained for a considerable period of time, and iii) SARS-CoV-2 can robustly replicate through multiple rounds of infection. Among easily accessible tissues, we selected the tonsils for establishment of human organoids and infection with SARS-CoV-2, as they have the potential to satisfy the unmet needs described above. From a clinical point of view, tonsils are also appropriate source material due to their susceptibility to diverse respiratory viruses including influenza virus, respiratory syncytial virus, and even coronavirus [22][23][24].
The tonsils are two oval-shaped immunologic organs in the back of the oral cavity [24] (Fig. S1A). Histologically, they are composed of mucosa-lining epithelium and underlying parenchymal B or T cell lymphocytes [24]. Multilayered, stratified tonsil epithelium are covered with differentiated epithelial cells at the superficial layer, which are generated from renewable stem-like epithelial cells in the bottom layer [25]. Unfortunately, a tonsil organoid model has not yet been established due to the difficulty of culturing tonsillar epithelial cells that can be stably maintained over long-term serial passage [25]. In addition, small laboratory animals, such as mouse and hamster, are not available for optimization of organoid construction because they lack orthologous organs [26]. However, establishment of an organoid model from tonsil tissues was expected to be achievable by applying advanced technologies for the long-term 3D culture of epithelial stem cells from human-derived specimens [27,28]. This approach might be valuable in efforts to understand cellular responses to viral infection and antiviral activity, as organoids are composed of diverse differentiated or proliferating cell types and can reconstitute the physiological conditions of human organs [29][30][31][32][33][34][35]. In this study, we sought to establish tonsil epithelial cell-derived organoids with morphological and molecular biological features comparable to those of epithelium of human tonsil tissues, and to examine their feasibility as an ex vivo model for SARS-CoV-2 infection.

Establishment of human tonsil epithelial organoids
To generate organoids from human tonsil epithelium, enzymatically dissociated cells from tonsil tissues were embedded in Matrigel. According to previous reports, they were cultured either in human colon organoid medium (HCM) or human prostate organoid medium (HPM) supplemented with growth factors including Noggin, fibroblast growth factor 2 (FGF2), FGF10, and R-spondin-1 (RSPO1), which have been identified to play pivotal roles in epithelial stem cell differentiation and proliferation ( Fig. S1A and Table S1) [36,37]. HPM yielded 2-fold more 3D organoids within 10 days than HCM (Fig. S1B, C). To achieve more efficient organoid formation, the composition of the medium was optimized by seeding dissociated tonsil epithelial cells in various conditioned HPM that contained or omitted individual components associated with cell proliferation. The largest organoids appeared with the highest organoid-forming efficiency when cultured in HPM in the absence of epidermal growth factor (EGF), which was subsequently defined as tonsil organoid formation medium (TfM) (Fig. 1A and B, and Fig. S1D). Microscopic and histological analyses revealed that organoids cultured in TfM developed stratified squamous epithelial structures resembling those of tonsil tissues between days 10 and 15 ( Fig. S2A and B). However, TfM limited subculture to two passages, and the cells were not available for additional expansion (Fig. S2C). To resolve this issue, we further modified TfM by removing SB202190, a selective p38 MAPK inhibitor, but by adding hepatocyte growth factor (HGF). These changes did not affect organoid size at passage 1, but well-shaped organoids were successfully expanded beyond five passages for 60 days. This optimized medium was defined as tonsil organoid expansion medium (TeM) ( Fig. 1C and D; Fig. S2D and E). Moreover, it did not affect reproducibility in the organoid formation after thawing of their cryopreservation (Fig. S3).
It has been reported that epithelial cell adhesion molecule (EpCAM), a representative marker for epithelial cells, is expressed in the basal layer of both epithelium of several tissues, including tonsil, and diverse epithelial organoids (Fig. S4A) [38][39][40][41][42]. To investigate whether the tonsil organoids originated from EpCAM + cells, we compared organoid formation ability of different cell populations from tonsil tissues on the basis of EpCAM expression. Flow cytometric analysis revealed that in contrast to EpCAM − cells, EpCAM + cells facilitated robust organoid formation ( Fig. 1E and F). This result suggested that tonsil organoids cultured in TeM are generated from EpCAM + cells, verifying their epithelium-like property.
Among CD44, integrin alpha 6 (ITGA6) and nerve growth factor receptor (NGFR), that are abundantly expressed in stem cells and involved in other organoid formation, we analyzed their effects on tonsil epithelial organoid formation [43][44][45][46]. Each subpopulation was isolated from E-cadherin + cells by flow cytometric cell sorting and then cultured in TeM (Fig. S5). The number of resulting organoids showed that it was highest in NGFR ++ cells, followed by NGFR + cells, ITGA6 ++ cells, CD44 ++ cells in that order. On the other hand, few organoids were obtained in the absence of any of these factors or in the lower level of CD44 (CD44 + ) or ITGA (ITGA + ) ( Fig. 1G and H). Cell sorting analysis indicated that expression of the three molecules, CD44, ITGA6 and NGFR, are required for differentiation and maturation of tonsil epithelial cells into organoids.

Histological and immunohistological characterization of human tonsil epithelial organoids
To determine whether the tonsil organoids could recapitulate the human original tonsil tissues as depicted in Supplementary Figure S4A, we explored their structural features and biomarker distribution. Staining of histological sections with hematoxylin and eosin (H&E), Alcian blue, periodic acid-Schiff (PAS), and Masson's trichrome revealed circular stratified squamous organoids with a biochemical composition similar to that of tonsil tissues ( Fig. 2A). In detail, we compared histochemical composition of multilayered cellular components in the tonsil organoids during their maturation with those of tissue epithelium either from tonsil surface or from crypt. The results revealed that basal layer cells of tonsil surface or crypt epithelium expressing NGFR, ITGA6, or CD44 are localized on the outer side of the organoids between days 10 and 15, whereas suprabasal or superficial layer cells expressing MUC1 were mainly detected in the interior of tonsil organoids (Fig. 2B). In addition, E-cadherin, a ubiquitous epithelial cell marker, was evenly distributed, whereas Ki67, a proliferation marker, (caption on next page) H.K. Kim et al. was predominantly expressed at the spherical boundary within the organoids, indicating active cell proliferation in that zone. These histological data suggested that tonsil organoid maturation was successfully achieved from a single cell of tonsil tissue, yielding a highly ordered stratified epithelium within 15 days in TeM.
The systematic organization of different cell types was further examined by immunofluorescence microscopy. For immunohistological experiments, organoids as well as tonsil tissues were co-stained with antibodies against specific markers representing individual tissue layers. First, we sought to determine the distribution of epithelial cells expressing MUC1, CK4, NGFR, CK5 and CD44 by using E-cadherin as a marker for the overall area of the epithelium. As expected, both MUC1and CK4-expressing cells were detected on the inner sides of organoids, which correspond to the suprabasal/superfical layers of the tissues (Fig. 2C), whereas NGFR-, CK5-, and CD44-expressing cells were detected on the outer ring of organoids, which corresponds to the basal layer of the tissue (Fig. S4B). We further examined the time course of tonsil organoid maturation, where Ki67 was used as a cell proliferation marker. Consistent with the immunohistochemistry data before (Fig. 2C, Fig. S4B), basal layer markers of tonsil tissue, including NGFR and CD44, as well as the suprabasal/superficial layer markers, including MUC1 and CK4, accumulated at the outer side and the inner side, respectively, completing maturation between days 10 and 15 (Fig. 2D). Importantly, colocalization of NGFR or CD44 with Ki67 in the organoids revealed that the essential factors required for tonsil organoid formation ( Fig. 1G and H) are continuously expressed at the proliferating cells throughout the differentiation and maturation period. In addition, a tonsil crypt-specific marker, cytokeratin 7 (CK7), was expressed at the inner layer cells of the mature organoids, whereas proliferating cell nuclear antigen (PCNA) was expressed in the outer border cells (Fig. S4C). Given this finding, it is assumed that the organoids might be differentiated along the crypt-surface axis by placing crypt-derived cells at the less proliferative inner space [47]. In line with the biomarker-based histological analyses, electron micrographs of the organoids at day 15 revealed typical ultrastructural features of stratified squamous epithelium, with characteristic elements such as keratohyalin granules and desmosome-tonofilament complexes [48] (Fig. 2E). Collectively, the histological, immunohistological and ultramicroscopic images strongly suggested that the outer and inner domains of tonsil organoids recapitulate the basal and suprabasal/superficial layers of the primary tonsil epithelium, respectively.

Gene expression profiles in human tonsil epithelial organoids and their response to a pathogen-derived immune stimulant
We investigated how similar the mRNA transcriptome of tonsil epithelial organoids was to that of tonsil tissues by comparing wholegenome expression levels of tonsil organoids from two donors (nos. 21-33 and 21-36) with their paired tissues, i.e., tonsil crypt and surface samples separately (Fig. S6). Heatmap analysis and hierarchical clustering of the high-throughput data from 21,448 genes showed high correlation coefficients between crypt and surface samples from a same donor (r = 0.96 for donor 21-33; r = 0.97 for donor 21-36) ( Fig. 3A and B). Even between the two donors, this gene expression profile was well correlated, showing r values above 0.91. It was interesting that the tonsil organoids showed donor-independent homogeneity (r = 0.98) but with relatively reduced correlation coefficient values, ranging from 0.75 to 0.80, to the tissue samples. The results suggested that gene expression profile became further synchronized when tonsil epithelial cells are matured into organoids.
Although overall gene expression of tonsil organoids was overlapped with that of tissues, we wondered which genes are differently expressed in organoids. Principal component analysis (PCA) plot showed that mRNA expression of the majority of genes (PC1, 88%) was similar between organoids and tissues (Fig. 3C). However, in a second major population of genes (PC2, 8%), organoids had a distinctive cluster from crypt and surface tissue samples. Specifically, 526 genes were upregulated but 796 genes were downregulated in organoids when compared to tissues (Fig. 3D). Gene ontology and pathway analysis revealed that metabolic reprogramming occurred in the organoids by stimulating ATP synthesis, tricarboxylic acid cycle (TCA) and cholesterol synthesis and in parallel, cell-cell communication was increased possibly for 3D morphogenesis (Fig. 3E, upper) [49]. In contrast, immune system was suppressed by downregulating IL-2 signaling pathway, antigen-activated B-cell receptor generation and cell adhesion molecules (Fig. 3E, lower). This inhibition might be related to biased differentiation of tonsil organoids to epithelial cells rather than to immune cells in the optimized media, TeM.
As a preliminary functional study, we examined how the organoids' innate immunity responds to a pathogen-derived molecule. It has been elucidated that microbial challenge stimulates tonsil epithelium to secrete C-X-C motif chemokines, including CXC ligand 1 (CXCL1) [50]. After treatment of organoids with lipopolysaccharide (LPS), a cell wall component from Gram-negative bacteria that binds to Toll-like receptor 4 (TLR4), we quantified several secretory cytokines. Culture supernatants of mock-or LPS-treated organoids were compared using membrane-based cytokine array (Fig. S7). Quantification of band intensity of the immunoblots revealed that interleukin-1 alpha (IL-1α), CXCL1 and IL-8 were significantly enhanced in the culture supernatants at 24 h after LPS treatment ( Fig. 4A and B). We then tested whether secretion of the chemokines or cytokines from tonsil organoids could affect cell migration. Counting of migrated cell number revealed that the migration index of HL-60 cells increased by approximately 2.6-fold when they were exposed to conditioned medium harvested from LPS-treated organoids compared to LPS-untreated organoids (Fig. 4C). As expected, mock-cultured medium without organoids had no effect on cell migration ability, irrespective of LPS treatment. The results are consistent with those of tissue samples from patients with tonsillitis, in which surface and crypt epithelium contributes to inflammatory processes as a defense mechanism for restricting microbial attack through secretion of chemokines as well as through facilitating neutrophil migration [50]. Our data confirms that the tonsil epithelial organoids are able to deploy their innate immune machinery in the presence of a TLR4-stimulating pathogen-associated molecular pattern (PAMP), LPS, in a similar way as tonsil tissues.
Angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin have been identified to be essential for SARS-CoV-2 viral entry to the target cells [51][52][53]. Specifically, ACE2 as a receptor binds to the viral spike protein, while TMPRSS2 and furin are involved in spike protein maturation for membrane fusion. Before investigating susceptibility of tonsil organoids to SARS-CoV-2 infection, it was necessary to determine whether these receptor or co-receptor proteins are expressed in the organoids. Immunofluorescence analysis, in which tonsil tissues were used as a control, exhibited that ACE2 is localized on the outer side, consisting of NGFR-positive basal layer cells, but less on the inner side, which consisted of MUC1-expressing suprabasal/superficial layer cellular components (Fig. 4D). In contrast, TMPRSS2 and furin showed relatively homogenous expression throughout the whole area of organoid sections. Time-course mRNA expression data from three different donors revealed that TMPRSS2 mRNA was upregulated between days 10 and 15; by contrast, ACE2 or Furin mRNA was comparably expressed for 15 days (Fig. 4E). Accordingly, it can be expected that tonsil organoids could be susceptible to SARS-CoV-2 infection.

SARS-CoV-2 infection of human tonsil epithelial organoids
To determine whether the tonsil epithelial organoids are susceptible to SARS-CoV-2 infection, the organoids after 7 days were incubated with SARS-CoV-2 at an MOI of 0.1 or 1, in which mock infections were performed as a negative control to exclude the possibility of donor-derived viral infection. Viral protein expression and RNA replication were monitored at day 2 post-infection by probing with anti-spike protein and anti-dsRNA antibodies, respectively, by co-staining the SARS-CoV-2 receptor, ACE2. As the viral infection visualized in human lung epithelial Calu-3 cells (Fig. S8), fluorescence microscopy images from the tonsil organoids revealed viral spike protein (Fig. 5A) and dsRNA (Fig. 5B) in the ACE2-expressing layers at both viral titers. Interestingly, ACE2 distribution was expanded from the basal layer into the suprabasal/superficial layer as SARS-CoV-2 robustly replicated.
As a next step, we assessed release of amplified viral particles from infected cells by quantification of viral RNA copies and determination of 50% tissue culture infective dose (TCID 50 ). Quantitative RT-PCR revealed a time-dependent increase in viral RNA levels, which was greater at 1 MOI than 0.1 MOI (Fig. 5C). Consistent with this result, an increase in infectious viral titer was observed 72 h after the lower-titer challenge but at 24, 48, and 72 h after the higher-titer challenge (Fig. 5D). This viral amplification was further verified in an independent experiment using three additional tonsil organoids established from different donors ( Fig. 5E and F). Even given the variation in susceptibility among donors, efficiency of SARS-CoV-2 replication increased continuously during 72 h post-infection. To visualize the generation of progeny viral particles at the final stage of the virus life cycle, we performed transmission electron microscopy (TEM) on SARS-CoV-2-infected tonsil epithelial organoids at 3 and 72 h post-infection at an MOI of 0.1. We observed a high density of amplified viral particles with the typical crown-like appearance on the external surface of the apical membrane in the SARS-CoV-2-infected organoids at 72 h, but not in mock-infected or 3 h post-infection samples (Fig. 5G). Viral diameter was distributed between 99.7 and 179.6 nm. Taken together, these results suggested that SARS-CoV-2 can infect the tonsil epithelial organoids, from which progeny viruses are released through viral protein expression and RNA-dependent RNA replication.

Activation of oncostatin M signaling and lipid metabolic remodeling but suppression of innate immune signaling by SARS-CoV-2 infection in the tonsil epithelial organoids
To perform Gene Ontology (GO) analysis affected by SARS-CoV-2 infection, the virus was highly purified by ultracentrifugation, that removes secretory molecules, such as pro-inflammatory cytokines, existing in the viral stock [54]. We compared mRNA expression levels of 21,449 genes of the organoids from three donors (21-3, 21-4 and 20-23) at 3, 24, and 72 h after infection. Their infection with SARS-CoV-2 was quality-controlled by qRT-PCR using the culture supernatant (Fig. S9). PCA plot showed that gene expression profile was similar in all donors before infection (Mock), but it became discriminated in a donor-and also time-dependent manner, with a greatest deviation in the donor 21-4 at 72 h post-infection (Fig. 6A). Among the analyzed genes, 395 genes were upregulated, which are involved in oncostatin M signaling, lysosomal pathway, iron transport and lipid metabolism (Fig. 6B-D). Their upregulation at the transcriptional level was confirmed by qRT-PCR of ANXA9, LOR, ATP6V1C2 and SPTLC3 mRNA, representatively (Fig. 6E). It is worthy of note that oncostatin M signaling pathway is highly activated in all virus-infected organoids. Given that this signaling was also stimulated in the tonsil organoids compared to their epithelial tissues (Fig. 3E), it seems that SARS-CoV-2 infection intensively coordinates cellular differentiation or proliferation, subsequently supporting its robust amplification there [55,56]. It was found that mRNA transcripts responsible for lipidomic remodeling are systematically upregulated, potentially being resulted from increased utilization of membrane fusion, lipid rafts, lipid droplets, endosomes and exosomes during the virus life cycle [57].
In contrast, 777 genes were markedly decreased at 72 h postinfection in the infected organoids (Fig. 6F). They are responsible for brain-derived neurotrophic factor (BDNF) signaling, cell growth or innate immune responses ( Fig. 6G and H). Quantitative RT-PCR analysis of ATF3, JUN, RSG2 and HSPA6 mRNA showed their time-dependent decreases, ensuring reliability of the GO analysis ( Fig. 6G and I). In accordance with a recent publication that reported a correlation between low serum BDNF level and severity of SARS-CoV-2 infection in patients [58], its signaling pathway was significantly suppressed in our organoid system. Overall, the transcriptome analysis suggested that SARS-CoV-2 infects the tonsil epithelial organoids and robustly self-amplifies there by remodeling cellular lipid metabolism but by suppressing host defense mechanism such innate immunity or growth factor signaling. This immunocompromised or immunosuppressed property of tonsil epithelial organoids acquired by viral infection might Fig. 2. Histological, immunohistological and ultrastructural analyses of tonsil epithelial organoids. (A) Histology of human tonsil epithelium tissues and organoids on day 15. Each sample was stained with H&E for morphological analysis, Alcian blue to visualize the mucosal layer and mucous-producing goblet cells, PAS to visualize mucosubstances, and Masson's trichrome to visualize connective matrix. (B) Immunohistological analyses of surface and crypt areas of human tonsil epithelium tissues along with tonsil organoids on days 5, 10 and 15. Each sample was immuno-stained with antibodies specific for epithelium markers, including NGFR, ITGA6, CD44, MUC1, and E-cadherin, as well as a cell proliferation marker, Ki67. (C) Immunofluorescence to compare distribution of suprabasal/superficial layer markers, MUC1 and CK4 (green), between the tonsil tissues and the organoids on day 15; the epithelial marker E-cadherin (red), was used as a control. Merged images with Hoechst 33342 staining (blue) are shown in the right-hand panels. (D) Immunofluorescence images showing a time course of organoid development on days 5, 10, and 15. Representative markers (green) for basal layer cells (NGFR and CD44; upper panels) and suprabasal/superficial layer cells (MUC1 and CK4; lower panels) were labeled with the corresponding antibodies; Ki67 (red) was used as a control for active proliferation. Merged images with Hoechst 33342 staining (blue) are shown in the right-hand panels. be related to its role as a reservoir for diverse viral infection or latency [59].

Evaluation of antiviral activity of remdesivir against SARS-CoV-2 infection in the tonsil epithelial organoids
To examine whether SARS-CoV-2 infection could be inhibited by an antiviral compound in the organoids, we treated virus-infected samples with increasing concentrations of remdesivir, a prodrug nucleoside analog that inhibits viral RNA replication. On day 2 after treatment, a dose-dependent reduction in viral RNA level as well as viral spike and nucleocapsid protein levels was observed in dose-dependent manner ( Fig. 7A-C). To test its reliability, tonsil epithelial organoids from three different donors (21-26, 21-27 and 21-33) were additionally prepared. Cell viability test by microscopically counting live and dead cells revealed that neither SARS-CoV-2 at an MOI of 0.1 nor remdesivir at a maximum dose of 100 μM did affect cell viability at day 2 post-infection (Fig. 7D). Dose response analysis by measuring viral RNA in the culture supernatants exhibited antiviral activity of remdesivir in all SARS-CoV-2-infected organoids with half-maximal effective concentration (EC 50 ) values ranging from 0.03 to 0.08 μM (Fig. 7E). These values have more improved antiviral activity when compared to those in our cell culture system, where it had EC 50 values of 35.4 μM in Vero 81 cells and 0.1-1 μM in Calu-3 cells [60]. This difference might be caused by variations in antiviral assay methods or viral growth kinetics in the cell types used. Confocal microscopy decisively visualized reduction of viral protein expression by remdesivir in the SARS-CoV-2-infected tonsil organoid (Fig. 7E). Our data emphasize that the tonsil epithelial organoids are competent for evaluation of the antiviral efficacy of small molecules before they are translated to clinical trials.

Discussion
The oropharynx, located behind the oral cavity and beneath the nasopharynx, functions in digestion and respiration. It consists of four parts: the tonsils, the base of the tongue, the soft palate, and the posterior oropharyngeal wall. The tonsils (also called the palatine tonsils or the oropharyngeal tonsils) contain large populations of immune and epithelial cells. This cellular diversity could account for the tonsils' multifunctional roles in front-line defense against bacteria or viruses invading the nasal or oral cavity, as well as in their function as reservoirs for infection by pathogens including Epstein-Barr virus, adenoviruses, influenza A and B viruses, herpes simplex virus, rhinovirus, enterovirus, and even human papillomavirus [61][62][63]. The two major proteins required for SARS-CoV-2 entry, ACE2 and TMPRSS2, have been reported to be expressed in the squamous epithelium lining oropharyngeal tonsillar tissue [64]. This motivated us to determine whether tonsil tissue is susceptible to the SARS-CoV-2 infection. However, the lack of a culture system for tonsillar epithelial cells has deterred in-depth cytopathologic or virological studies of the pandemic pathogen. In the present study, we were able to establish a reliable culture system for human tonsil epithelial organoids by optimizing culture medium composition, thereby recapitulating the dominant features observed in in vivo tonsil epithelium, such as stratified squamous epithelial layers and appropriate distributions of tonsillar biomarkers (Figs. 1 and 2). The major population of genes (88%) of the organoids follow a transcriptome profile of tonsil epithelial tissues, both crypt and surface (Fig. 3). The tonsil epithelial organoids expressed the major components, such as ACE2, TMPRSS2 and furin, which are essential for viral attachment and fusion during the entry step, and were highly susceptible to SARS-CoV-2 infection (Figs. 4 and 5). Consequently, the virus was vigorously amplified through multiple rounds of infection, resulting in abundant secretion of progeny viral particles. Our findings suggest that the tonsil epithelium could be one among the major target organs for primary infection of SARS-CoV-2, and that its organoids are a desirable ex vivo model for investigation of viral susceptibility or transmissibility.
'Cytokine storm' is an initial cause of acute respiratory distress syndrome (ARDS), multi-organ failure, and severe pneumonia, which result in considerably mortality among COVID-19 patients [65]. This hyperactive inflammatory response is primarily associated with abrupt release of pro-inflammatory or inflammatory mediators, including IL-6, TNF-α, and CXCL8 (also named IL-8) [1]. However, SARS-CoV-2 infection does not trigger 'cytokine storm' in children (a mean age of 7.3 year) with COVID-19, not affecting the levels of plasma pro-inflammatory cytokines [66]. From these contradictory findings, our cDNA microarray analysis stood for the latter by showing that SARS-CoV-2 infection suppresses NF-κB-related pro-inflammatory cytokine pathways in the tonsil epithelial organoid at 72 h post-infection (Fig. 6F-I). Thus, we assume that among the upper respiratory tract tissues, the tonsil epithelium might be an optimized place for SARS-CoV-2 infection, through which they could play a pivotal role as the antigen presenting cells in the circumstances with abundant immune cells. Recently, preparation of immune organoids from tonsils and their remodeling adaptive immunity have been established by L. E. Wager et al. [67]. As a further study combining this technology, we are going to explore how the adaptive immunity is educated and lipid metabolic reprogramming changes when tonsil immune cell-derived organoids are co-cultured with SARS-CoV-2-infected autologous epithelial organoids. This approach could contribute to elucidating immunological tripartite crosstalk between tonsil epithelial cells, immune cells, and SARS-CoV-2, providing a comprehensive understanding not only of the host defense mechanism from the standpoint of both innate and adaptive immunity, but also of viral immune evasion strategies counteracting host immunity.
We evaluated the therapeutic efficacy of remdesivir in the tonsil organoid model, demonstrating its value as a drug candidate screening platform for development of novel SARS-CoV-2 therapies. This organoid system has advantages as an alternative to animal models, particularly when they target host factors involved in the viral life cycle or immune regulation. It is nontrivial because the efficacy of those antivirals could be underestimated (or impossible to assess) in animals due to low genetic homology or null function in model organisms. This is the first study to report the generation of human tonsil epithelial organoids and their suitability for infection by a respiratory virus, especially SARS-CoV-2. From the clinical perspective, tonsils are a readily accessible intraoral organ from which specimens are frequently underutilized after surgery; by contrast, obtaining samples from the kidney, liver, bronchus, or gut requires invasive and aggressive procedures. Thus, tonsil epithelial organoids can be suggested as an attractive ex vivo model for fundamental or applied research on infections by SARS-CoV-2 and potentially other pathogenic microbes.

Clonal organoid formation assay from cells isolated by fluorescenceactivated cell sorting (FACS)
To determine the origin of cells forming the organoids, cells dissociated from tissues as mentioned above were stained with anti-Ecadherin (cat no.  (Table S2). They were individually sorted using an S3e Cell Sorter (Bio-Rad, Hercules, CA, USA) and cultured in Matrigel with TeM for 15 days.

SARS-CoV-2 amplification and purification
SARS-CoV-2 (BetaCoV/Korea/KCDC03/2020) provided by the Korea Disease Control and Prevention Agency (Cheongju, Korea) was amplified in Vero E6 cells (American Type Culture Collection, Rockville, MD, USA) through three passages in DMEM (HyClone, South Logan, UT, USA). After centrifugation of the supernatants at 3000 g for 10 min, the viral stock was stored at − 80 • C before use.
For host transcriptome analysis, SARS-CoV-2 was purified through ultracentrifugation according to a previous report with some modifications [68]. Briefly, the virus stock was loaded onto 20% sucrose dissolved in TNE buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl and 1 mM EDTA) and centrifuged at 175,000 g for 3 h at 4 • C using Optima XPN-100 Ultracentrifuge with SW 32 Ti rotor (Beckman Coulter, Brea, CA, USA). The viral pellet was resuspended in PBS (1/2 of the starting volume). Viral titer was determined by plaque assay before use. All experiments with infectious SARS-CoV-2 were performed within a Biosafety Level 3 (BL3) facility in KRICT.

Infection of tonsil organoids with SARS-CoV-2
Tonsil epithelial organoids containing 6 × 10 4 cells at day 7 of passage 2 were suspended at 37 • C for 1 h in 5 μL TeM for infection with the same volume of SARS-CoV-2 at an MOI of 0.1 or 1. Unadsorbed virus was removed by washing twice with 1 mL of advanced DMEM/F12 with centrifugation at 1000 g for 10 s. Each sample of mock-infected or SARS-CoV-2-infected organoids was embedded in 20 μL Matrigel on a 48-well plate and cultured in 300 μL TeM supplemented with 10 μM Y-27632.
The supernatants were harvested at 3, 24, 48, and 72 h post-infection for viral RNA quantification or infectious viral titration. For cDNA microarray analysis, cell lysates were harvested for RNA extraction.

Histology, immunohistochemistry, and immunofluorescence
Tissues and organoids were fixed in 4% paraformaldehyde (PFA; Bio Solution, Suwon, Korea) and embedded in paraffin. Paraffin sections (5 μm thick) were deparaffinized in xylene and hydrated in a graded series of ethanol. They were then stained using H&E, Alcian blue (Agilent Technologies, Santa Clara, CA, USA), PAS staining kit (Abcam, Cambridge, MA, USA), or Masson's trichrome staining kit (Dako, Carpinteria, CA, USA). For immunohistochemistry, antigen retrieval was performed by incubating in sodium citrate buffer (10 mM sodium citrate with 0.05% Tween 20, pH 6.0) at 95 • C for 30 min. Endogenous peroxidase was blocked by incubating in 3% H 2 O 2 in methanol for 10 min. After blocking with 1% BSA in PBS, sections were incubated at 4 • C Fig. 4. Innate immune response and SARS-CoV-2 (co)receptor expression in tonsil epithelial organoids. (A) Human cytokine array for secreted proteins from the tonsil organoids before (− ) and after (+) LPS treatment at a concentration of 10 μg/mL for 24 h. (B) Changes in cytokine secretion levels were determined by densitometric analysis of the blots shown in (A). Values relative to the control levels (-LPS) are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by two-way ANOVA with Sidak's pairwise multiple comparison test. ***, p < 0.001; ****, p < 0.0001. n.d., not detected. A.U., arbitrary units. (C) Effect of LPS-stimulated culture supernatants on cell migration. HL-60 cells were treated with mock-or tonsil organoid culture medium before (-LPS) and after LPS stimulation (+LPS). Numbers of migrated cells are presented as means ± SEM of the three independent experiments. Two-way ANOVA with Sidak's multiple comparison test was used for statistical analysis. n.s., not significant. *, p < 0.05. (D) Immunofluorescence staining of ACE2 (left), TMPRSS2 (middle) and furin (right) (red) expressed in tonsil organoids on day 15 as well as in tonsil tissues. NGFR and MUC1 (green) are stained as epithelial markers for the basal and suprabasal/superficial layers, respectively. Merged images with Hoechst 33342 staining (blue) are presented on the right side of each panel. overnight with primary antibody and then incubated at room temperature for 30 min with a biotinylated secondary antibody (anti-mouse IgG or anti-rabbit IgG) using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were stained according to the avidin-biotin complex method using the Vectastain DAB kit (Vector Laboratories).
For immunofluorescence analysis, fixed samples were cryoprotected by immersion in PBS containing 30% sucrose and 0.1% sodium azide at 4 • C, and then embedded in optimal cutting temperature (OCT) compound (Sakura-Finetek, Torrance, CA, USA). Rapidly frozen sections (4 μm thick) were pre-blocked with 5% normal horse serum (Vector Laboratories) in PBS at room temperature for 2 h, followed by incubation at 4 • C overnight with primary antibodies specific for cellular biomarkers, viral spike protein, or dsRNA. They were then incubated with fluorescently-labeled secondary antibodies at room temperature for additional 2 h. Nuclei were counterstained with Hoechst 33342 (1 μg/ mL; Sigma). Acquisition of confocal images was performed on an LSM 880 confocal microscope (Carl Zeiss, Jena, Germany). Information about antibodies used in immunofluorescence analysis is summarized in Table S2.

Transmission electron microscopy (TEM)
To observe ultrastructure of tonsil organoids or release of progeny SARS-CoV-2 particles from infected organoids, TEM analysis was performed according to a previous report with some modifications [69]. Briefly, tonsil organoids were fixed in Karnovsky's fixative solution (2% glutaraldehyde, 2% paraformaldehyde, 0.5% CaCl 2 in 0.1 M phosphate buffer, pH 7.4) and embedded in the epoxy resin after post-fixation with 1% OsO 4 dissolved in 0.1 M phosphate buffer. Ultrathin sections were processed using an ultramicrotome (EM UC7; Leica, Wetzlar, Germany) and placed on a copper grid stained with 6% uranyl and lead citrate. The samples were observed using a JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan).

Microarray analysis
To analyze the genetic similarity between tonsil tissues, crypt or surface, and organoids or whole-genome expression changes resulting from SARS-CoV-2 infection, total RNA was extracted from tissue and organoids using the MagListo 5 M Cell Total RNA Extraction Kit (Bioneer, Daejeon, Korea) or Trizol reagent (Invitrogen). mRNA was reversetranscribed into cDNA using GeneChip Whole Transcript (WT) cDNA Synthesis and Amplification kit (Affymetrix, Santa Clara, CA, USA). After fragmentation of cDNA, it was biotin-labeled using the GeneChip WT Terminal labeling kit (Affymetrix) and hybridized to the Affymetrix Gene Chip Array (Affymetrix) at 45 • C for 16 h. Sample quality and gene expression profiling were certified at Macrogen (Seoul, Korea). To assess global gene expression profiles, we performed clustering analysis based on the centered correlation coefficient between transcriptomes of different samples. Gene set enrichment was analyzed using the Ingenuity Pathway Analysis software (IPA; Qiagen, Hilden, Germany).
To evaluate gene expression changes observed in microarray analysis, target mRNA expression was quantified by qRT-PCR. Total RNA was reverse-transcribed with random hexameric oligonucleotide primers using SuperScript III First-Strand Synthesis System (Invitrogen) and cDNA was amplified with target-specific primers and SYBR Green Master Mix the CFX96 (Bio-Rad) using Touch real-time PCR instrument (Bio-Rad). Their expression was normalized to GAPDH gene. All primer sequences used in this study are listed in Table S3.

Cytokine array and chemotaxis assay
The cytokine profile assay was performed using the Proteome Profiler Human Cytokine Array kit (R&D Systems, Minneapolis, MN, USA). Culture supernatants from LPS-treated (Sigma) or -untreated tonsil organoids were collected and incubated with precoated Proteome Profiler array membrane at 4 • C overnight. The membrane was then washed and incubated with streptavidin-horseradish peroxidase (HRP) buffer for 30 min. After treatment with Chemi Reagent Mix (provided in the kit), dot densities were imaged on an LAS4000 (GE Healthcare, Chicago, IL, USA) and quantified using the ImageJ software (https://imagej.nih. gov/ij/). To perform the chemotaxis assay, HL-60 cells (Korean Cell Line Bank, Seoul, Korea) were differentiated into granulocytes by culture in the presence of 1.3% DMSO (Duchefa Biochemie, Haarlem, The Netherlands) for 7 days, as described previously [70]. Cell monolayers grown on Transwell inserts (Corning) at a density of 5 × 10 5 cells/well in RPMI 1640 medium (Gibco) containing 0.5% BSA were exposed to supernatants that were collected from LPS-treated or -untreated organoids. After 1 h, migrated HL-60 cells were counted using a LUNA Automated Cell Counter (Logos Biosystems, Annandale, VA, USA).

Viral RNA quantification and TCID 50 determination
Viral RNA was purified from the culture supernatant at the indicated time points using Viral RNA Purification Kit (Qiagen). One-step RT-PCR targeting the viral nucleocapsid (N) gene was performed using a diagnostic kit (PCL Inc., Seoul, Korea) and the CFX96 Touch real-time PCR instrument (Bio-Rad). In all experimental sets, RNAs purified from serial dilutions of a virus stock with quantified plaque titers were used as a standard to determine the absolute viral RNA copy number in the samples. In parallel, Vero CCL-81 cells were seeded at a density of 2 × 10 4 cells per well in 96-well plates and then treated with serially-diluted culture supernatants from SARS-CoV-2-infected organoids at an MOI of 0.1 or 1. On day 2 post-infection, the cells were fixed and permeabilized with chilled acetone:methanol (1:3) solution at room temperature for 10 min. Viral spike (S) protein was probed using anti-S antibody (Genetex, Irvine, CA, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen) and cellular nuclei were stained with 4',6diamidino-2-phenylindole (Invitrogen) as described previously report [71]. Fluorescence images were captured and analyzed on an Operetta High-content Screening System (PerkinElmer, Waltham, MA, USA) and quantified using the built-in Harmony software. Compared with the control infection (100%), dilution folds resulting in 50% infection were calculated to determine the 50% tissue culture infectious doses (TCID 50 ). (green) and a cellular receptor, ACE2 (red), in tonsil epithelium organoids on day 3 after SARS-CoV-2 infection at MOI of 0 (Mock), 0.1, or 1. Scale bar is 100 μm.
Merged images with nuclei (blue) and their zooms are presented in the right panels. (C) Quantitative RT-PCR for detecting viral RNA in the culture supernatants of tonsil organoids infected with SARS-CoV-2 at an MOI of 0.1 or 1 at various time points after infection. (D) Determination of the amount of infectious viral particle in culture supernatants of SARS-CoV-2-infected tonsil organoids from the same donor of (C). Viral titers were measured by infection of fresh Vero CCL-81 cells with organoid culture medium for 2 days and by staining with anti-spike protein antibody, as described in Materials and Methods. (E, F) Both viral RNA copies (E) and infectious SARS-CoV-2 titers (F) in culture supernatants were determined independently from three additional tonsil organoids (donors 20-13, 20-14 and 2016). Twoway ANOVA with Dunnett's multiple comparison test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Antiviral activity of remdesivir against SARS-CoV-2 in tonsil organoids
Antiviral assays were performed according to our previous report with some modifications [60]. Briefly, 7-day-cultured tonsil organoids were infected with SARS-CoV-2 at an MOI of 0.1 and embedded in 20 μL of fresh TeM plus Matrigel (6 × 10 4 cells per well in 48-well plates). Remdesivir (purity 99.84%; MedChemExpress, Monmouth Junction, NJ, USA) was 10-fold serially diluted in TeM from 10 to 0.1 μM and used to treat SARS-CoV-2-infected organoids. For determining EC 50 values, which are chemical concentrations to reduce viral RNA titers by 50%, three-fold serial dilutions of remdisivir (from 100 μM to 1.7 nM) were treated to the SARS-CoV-2-infected organoids from three different donors in triplicate. On day 2, the culture supernatants were harvested to measure the amount of viral RNA by quantitative RT-PCR. In parallel, cell lysates were collected from four wells using RIPA buffer (LPS solution, Daejeon, Korea), and viral protein expression levels were compared by Western blot analysis. The immuno-transferred membrane was probed with anti-spike primary antibody (GeneTex, Irvine, CA, USA) or anti-nucleocapsid antibody (Sino Biological, Beijing, China) followed by HRP-conjugated anti-mouse goat IgG as the secondary antibody (Invitrogen).

Statistical analysis
Statistical significance of differences was analyzed using GraphPad Prism software package, version 8.4.3 (GraphPad, San Diego, CA, USA). Unpaired two-tailed t-tests were used for comparisons of two groups. In general, two-way ANOVA with Dunnett's or Sidak's multiple comparison tests were used to compare multiple groups, but if needed, one-way ANOVA was performed followed by Dunnett's or Tukey's multiples comparison tests. p < 0.05 was considered significant. Unless otherwise specified, all experiments were performed in triplicate and their results are presented as means ± SEM from three independent experiments.

Data availability
The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. presented as means ± SEM of four independent experiments using tonsil organoids from a single donor (no. . Statistical analysis was performed by ordinary one-way ANOVA with Dunnett's multiple comparison test. **, p < 0.01. (B) Western blot analysis showing reduction of viral spike and nucleocapsid proteins in two different organoids samples (donors 20-16 and 21-36, respectively) in the presence of remdesivir. Beta-actin was used as a loading control. (C) Reduction of nucleocapsid protein by remdesivir was quantified from Western blot analysis with three different organoid samples (donors 21-16, 21-34 and 21-36). Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparison test. ****, p < 0.0001. (D) Cell viability test. Organoids from three different donors (21-26, 21-27 and 22-33) were mock infected or infected with SARS-CoV-2 alone (MOI, 0.1) or in the presence of 100 μM remdesivir. On day 2, organoids from three different wells were dissociated with trypsin-EDTA and individually stained with trypan blue. Live and dead cells were counted by using an automated microscopic cell counter. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test. n.s., statistically not significant. (E) Organoid samples from three different donors (21-26, 21-27 and 22-33) were infected with SARS-CoV-2 (MOI, 0.1) and treated with increasing concentrations of remdesivir. On day 2 post-infection, RNA samples were harvested from culture supernatants for qRT-PCR against the viral nucleocapsid gene. EC 50 values are calculated in three independent experiments and expressed as mean ± SD. (F) Confocal miscopy showing reduction of viral spike protein by remdesivir. Tonsil epithelial organoids (donor 21-36) were mock-infected (upper) or infected with SARS-CoV-2 at an MOI of 0.1. The infected organoids were treated with DMSO (middle) or remdesivir (10 μM; lower) for 2 days. Viral spike protein (blue) and its binding receptor ACE2 (red) were stained with their specific antibodies. Nuclei (blue) were counter-stained with Hoechst 33342. Merged images and their zooms are displayed on the right side. Scale bar is 100 μm.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)