key: cord-0875627-86qrydlo
authors: Rai, Pallavi; Chuong, Christina; LeRoith, Tanya; Smyth, James W.; Panov, Julia; Levi, Moshe; Kehn-Hall, Kylene; Duggal, Nisha K.; Lucarelli, James-Weger
title: Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis
date: 2021-09-03
journal: Virology
DOI: 10.1016/j.virol.2021.08.014
sha: 9221119902b37baee7d91e0a81ecb77c036633cb
doc_id: 875627
cord_uid: 86qrydlo

The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.

Coronavirus disease-2019 is the third pandemic in the 21 st century caused by a novel 49 coronavirus, after severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 [1] [2] [3] [4] 

Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 [5] [6] [7] . COVID-19 is responsible for 51 ~191 million confirmed cases, with over 4 million deaths globally, and ~33 million confirmed cases and 52 ~600,000 deaths in the U.S. alone, as of July 21 st , 2021 [8] . COVID-19 is characterized by fever and 53 respiratory symptoms, which can progress to more severe and fatal disease [9] . Infection with SARS-54

CoV-2, the causative agent of COVID-19, is more likely to cause critical illness or deaths in the elderly, 55 J o u r n a l P r e -p r o o f and xylazine anesthesia, or RPMI-1640 media alone for mock-infected groups. Six infected and four 128 control mice from each group were sacrificed at days four and ten after SARS-CoV-2 infection. Mice 129 were weighed daily post-SARS-CoV-2 infection and monitored for visual symptoms of the disease. 130

Blood was collected via submandibular bleeds (~200 µL per mouse), serum was collected via 131 centrifugation at 5000 x g for 10 minutes, transferred to fresh tubes and stored at - 80°C. 132 Histopathology and organ titration 133 Tissues for viral titration were harvested aseptically in 2 mL tubes containing a 5 mm stainless steel bead, 134 RPMI-1640, 10 mM HEPES and 1% FBS (herein referred to as the "tissue diluent"), to a final 135 concentration of 10% weight by volume. Tissues were homogenized in a Tissuelyser II (Qiagen) at 30 136 cycles per second for 2 mins and then centrifuged at 5000 x g for 10 mins. Plaque assays were performed 137 on the clarified supernatant. Briefly, Vero E6 cells at 100% confluency were inoculated with 50 µL of the 138 serially diluted samples and incubated at 37°C in 5% CO2 for 1 hour. Overlay media containing 0.6% 139 tragacanth gum, 1x MEM (minimum essential media), 20 mM HEPES and 4% FBS were then added, and 140 the plates were allowed to incubate for two days for plaque formation. 141

For histopathology, the tissues harvested from mice were fixed in 4% paraformaldehyde for at 142 least one week. The Virginia Tech Animal Laboratory Services (ViTALS) performed paraffin embedding, 143 sectioning and hematoxylin-eosin staining, and a board-certified pathologist scored the slides in a blinded 144 manner. 145

A section of the infected lung tissues (4 dpi) was collected in 0.5 mL of TRIzol LS reagent 147 (ThermoFisher) in 2 mL tubes with a 5 mm bead. Lung tissues were homogenized using a TissueLyser II 148 (Qiagen) at 30 cycles per second for 2 mins and stored at -80°C. Samples were thawed and total RNA 149 was extracted using the manufacturer's protocol for TRIzol extraction. 150 J o u r n a l P r e -p r o o f

To quantify SARS-CoV-2 genomes, the 2019-nCoV RUO kit from Integrated DNA Technologies (IDT, 152 Leuven, Belgium) was utilized. N2 combined primer-probe mix from the kit was used (Table S2) with 153

Quantabio qScript XLT-One-Step RT-qPCR ToughMix (2X). The Bio-Rad CFX-96 (Hercules, CA, 154 USA) was used for RT-qPCRs with the following conditions: 50°C for 10 mins for reverse transcription, 155 95°C for 3 mins for initial denaturation and polymerase activation, followed by 45 cycles of 95°C for 10 156 secs for denaturation, and 60°C for 30 secs for annealing/extension. For generating the standard curve, we 157 used N gene RNA generated by in vitro transcription and used ten-fold serial dilutions to the point where 158 no genome was detectable by qPCR. The virus concentration was calculated by fitting the Cq values of 159 the samples to the standard curve and expressed in terms of N-gene copies/mL. 160

RT-qPCR was also performed on RNA extracted from the 4 dpi lung samples with NEB Luna 161

Universal One-Step RT-qPCR Kit with SYBR-Green (NEB, Ipswich, MA, USA) to quantify cytokines. 162

Primers were obtained from IDT and are listed in Table S2 . The conditions for the reactions in a Bio-Rad 163 CFX-96 were: 50°C for 10 mins for reverse transcription, 95°C for 1 min for initial denaturation and 164 polymerase activation, followed by 45 cycles of 95°C for 10 secs for denaturation, and 60°C for 30 secs 165 for annealing/extension, followed by a melt curve. The samples were normalized with mouse GAPDH as 166 the reference gene with respect to mock-infected groups (from the corresponding diet) as a control. The 167 fold change between the SARS-CoV-2 infected lean and obese mice was calculated using the Delta Delta 168 Ct (∆∆Ct) method of relative quantification [39] . were performed on data after testing them for normality using the Shapiro-Wilk test. 176

Wild type C57BL/6N mice fed a high fat diet had increased weight 178 gain and blood glucose levels.

The proportion of COVID-19 patients requiring intensive care was previously reported to be directly 180

proportional to the body-mass index (BMI), being highest in patients with BMI ≥35 (morbidly obese) 181

[40]. Obesity has also been correlated with severe outcomes during the H1N1 influenza A epidemic [17, 182 41-43] with SARS-CoV-2 exhibit worse disease outcomes as compared to their lean counterparts. 186

We observed a significant weight gain in high-fat diet (HFD)-fed mice compared to the low-fat 187 diet (LFD)-fed groups starting 7 weeks after the initiation of diets (p-value <0.0001) (Fig. 1A) . Weights 188 were analyzed on the combined data for males and females on LFD and HFD. Separate analysis for males 189 and females is presented in supplementary figure S1. To assess other metabolic parameters, we measured 190 overnight fasting blood glucose levels after 9 and 16 weeks of the initiation of diets. We tested only 10 191 mice per group at 9 weeks to reduce stress on the mice. Hyperglycemia, defined as overnight fasting 192 glucose levels of ≥150 mg/dL, was not significantly different between the lean and obese groups at 9 193 weeks (p-value=0.2344) (Fig. 1B) . By 16 weeks, however, there was a significant difference between 194 HFD and LFD groups (p-value=0.0001) (Fig. 1C) . This 15 to 20-week diet regimen produced a mouse 195 model that mirrored obesity and hyperglycemia in humans and, therefore, was appropriate for infection 

A) Four-week-old C57BL/6N mice (20/group) were fed a high-fat diet (60% fat) or a low-fat diet (10% fat) for 15-20 weeks.

Weights were measured weekly after initiating the diets (****p<0.0001, 2way-ANOVA with Sidak's multiple comparisons test).

B) Blood glucose levels were measured following 9 weeks of feeding (n=10) (p=0.2344, Mann-Whitney test). C) Glucose levels 202 were measured after 16 weeks of feeding (n=20) (***p=0.0001, Mann-Whitney test). The error bars indicate standard deviation 203 (SD) from mean.

Adenovirus-transduction caused morbidity in obese mice, 206 independent of SARS-CoV-2 infection.

Since mouse ACE2 receptors do not allow for WT SARS-CoV-2 infection, we first transduced mice with 208 a replication-defective adenovirus encoding for human ACE2 (AdV-hACE2) intranasally to achieve 209 hACE2 expression in the lungs and render mice susceptible to infection. We then inoculated the mice 210 with SARS-CoV-2 or diluent five days post-transduction, as previously described [30,31] ( Fig. 2A) . 211

Surprisingly, the obese mice inoculated only with AdV-hACE2 showed significant weight loss, similar to 212 those infected with both AdV-hACE2 and SARS-CoV-2 ( Fig. 2B ). No weight loss was observed in lean 213 mice treated with AdV-hACE2 or AdV-hACE2 and SARS-CoV-2. We found a significant weight loss in 214 obese-AdV only mice compared to the lean-AdV only group starting from days 5 (p-value=0.0010) 215 through day 9 post-AdV transduction (p-value=0.0242). The weights of the mice starting from the day of 216

AdV inoculation in both percent weight loss and grams are presented in supplementary figure S2. 217

Collectively, these data suggested that the AdV-hACE2 vector at the dose used here, produced morbidity 218 in obese mice. However, we proceeded to test other parameters to determine if differences could be found 219 between lean and obese mice in terms of viral replication, immune response, or lung pathology 220

To measure viral replication, a subset of the mice was euthanized on days 4 and 10 post-SARS-222

CoV-2 inoculation and infectious virus was quantified in the lung homogenates. As expected, mice 223

receiving only AdV-hACE2 tested negative for SARS-CoV-2 (Fig. 2C ). Infectious lung viral titers 224 between lean and obese infected groups were similar (p-value=0.7377) at 4 days post-SARS-CoV-2 225 infection, and 1 lean and 2 obese mice were negative for infectious virus. We next used RT-qPCR against 226 J o u r n a l P r e -p r o o f the N-gene as a more sensitive means to detect viral RNA. At 4 days post-SARS-CoV-2 inoculation, all 227 lean mice were positive for the N-gene, while both infectious-virus-negative obese mice were negative, 228

likely indicating a lack of productive infection. In Figure 2E , we present RT-qPCR data for the N-gene 229

for lungs collected at 10 days post-SARS-CoV-2 infection; all 6 lean mice and 5 of 6 obese mice tested 230 positive for SARS-CoV-2 N-gene, and all the mice were negative for infectious virus tested by plaque 231 assays (data not shown). No differences were observed between lean and obese groups in N-gene 232 copies/mL at either timepoint. This finding is consistent with previous studies that found no significant 233 difference in viral titers at peak viral replication between lean and obese mice infected with influenza 234

Severe COVID-19 disease is characterized by low levels of type I interferons (IFN-α and β) and 236 over-production of inflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor alpha 237 (TNF-α) [46] [47] [48] [49] [50] . Moreover, invasive mechanical ventilation (IMV) was associated with severe obesity in 238 patients infected with SARS-CoV-2, irrespective of age, sex, diabetes, and hypertension [40] . IL-10, IFN-β and IFNγ was measured by RT-qPCR. The relative expression of these cytokines was 246 calculated by the ∆∆Ct method, with hACE2-AdV infected groups as control and mouse GAPDH as the 247 reference gene. We selected these cytokines since they have previously been identified as predictors of 248 severe COVID-19 disease outcome [51] [52] [53] . We observed significantly higher expression of IFN-β (**p-249 value=0.0042) and IFNγ (*p-value=0.0376) in SARS-CoV-2 infected obese mice compared to their lean 250 infected counterparts at 4 dpi (Fig. 3A) . At 10 dpi, there was no significant difference in the expression of 251 J o u r n a l P r e -p r o o f IL-6, IL-10 and IFN-β, but IFNγ was significantly upregulated in obese mice infected with SARS-CoV-2 252 compared to lean infected mice (**p-value=0.0022) (Fig. 3B) 

IFNγ to be potent inhibitors of SARS-CoV-2 [54], which might explain why some obese mice did not 254 become infected following SARS-CoV-2 infection. 255

We also examined lungs, liver, and heart for histopathological lesions. Lungs were evaluated and 256 scored based on evidence of interstitial inflammation, intra-alveolar hemorrhage, and peribronchiolar and 257 perivascular lymphoid hyperplasia. There was no evidence of interstitial or alveolar septal necrosis or 258 hyaline membrane formation. Livers were evaluated for evidence of lipidosis and single cell necrosis. 259

The hearts were evaluated for evidence of myocardial necrosis and inflammation. Representative lung 260 images collected at 4 dpi are presented in Fig. 4A . The lung histopathology scores were similar for obese 261 mice inoculated with both AdV-hACE2 and SARS-CoV-2 compared to their AdV-hACE2 only 262 transduced counterparts at 4 dpi ( Fig. 4B ) but trended towards being more severe for the SARS-CoV-2 263 infected mice at 10 dpi (Fig. 4C) . Lung pathology scores were lower for SARS-CoV-2 infected lean mice 264 compared to their obese counterparts at 4 dpi (p-value= 0.03) but were similar at 10 dpi (p-value>0.9999). 265

We hypothesize that the early difference was due to IFN-β production in infected obese mice controlling 266 replication, and thus disease, but that lower IFN-β later in infection enabled SARS-CoV-2 replication and 267 produced the resulting lung pathology. No differences in the histopathology scores for heart and liver 268 were observed between the groups. Taken together, no major differences were observed between the two 269 groups in tissue pathology; however, these data are likely confounded by the morbidity observed in AdV-270 hACE2 alone treated obese mice. 271

The mechanisms underlying the morbidity observed in AdV-hACE2 transduced obese mice are 272 currently unclear. Future work using a control or empty AdV will enable parsing out of vector-related 273 mechanisms vs. ectopic expression of hACE2 will be informative. Replication-defective adenoviruses 274 have been used in many gene therapy applications [55, 56] and are known to activate both innate and 275 adaptive immune responses in humans [57] [58] [59] [60] . Furthermore, they have been shown to cause 276 hepatotoxicity in mice due to induction of TNF-α in the liver [61, 62] and dose-dependent morbidity in 277 non-human primates [63] . Accordingly, we posit that obese hosts may be more permissive to disease 278 following transduction with this dose of AdV-hACE2, possibly due to an overzealous immune response 279

to the vector and SARS-CoV-2, as shown by increased interferon gene expression. The data presented 280 here suggest some caution should be taken when using adenovirus vectors in broader populations. 281

Notably, the race for developing a vaccine against SARS-CoV-2 at pandemic speed [64] has renewed the 282 interest of researchers in using adenovirus vectors, which makes our findings worth considering. 283

The major limitation of our study was the use of the adenovirus vector for transduction of mice with the 285 hACE2 receptor. Inoculation of obese mice with adenovirus vector at this dose caused weight loss, 286 confounding our ability to assess obesity's impact on SARS-CoV-2 infection in mice, the main objective 287 of our study. We did not test a lower dose of the AdV-hACE2 vector, however, which may have provided 288 enough ACE2 expression without any off-target effects. Another limitation of this study was the lack of a 289 true mock-treated group, with no AdV-hACE2 or SARS-CoV-2 infection or an AdV with a control 290 transgene, in the absence of which we cannot quantify the impact of the adenovirus vector versus hACE2 291 transgene on the obese host. Moreover, these differences could be specific to C57BL/6N mice and we 292 might observe different results with other mouse strains; though, we were constrained to use C57BL/6N 293 mice as they are sensitive to DIO and are an ideal model for human obesity [65] . Another major limitation 294 was not testing a wide range of cytokines; thus, we cannot make generalized claims about inflammation. 295

Despite these constraints, this study provides useful information to the field relating to SARS-CoV-2 296 mouse models and advises caution when considering the AdV-hACE2 model as laid out here and 297 elsewhere for nutrition studies. 298

The increased risk of grave illness and fatalities in obese people post-SARS-CoV-2 infection underscores 300 the importance of having an effective animal model to study the mechanisms underlying worsened 301 disease outcomes. Here, we generated DIO mice and used a previously established adenoviral-302 transduction model to render them susceptible to SARS-CoV-2 infection. Our results demonstrate that 303 although adenovirus-transduction sensitized obese and lean mice to SARS-CoV-2 infection in their lungs, 304

it also induced morbidity in obese mice. Therefore, this model was not appropriate for studying the 305 relationship between underlying co-morbidities like obesity or diabetes and severe COVID-19. However, 306 optimization of the viral dose, the strain of mice used, and other parameters may reveal the utility of the 307 AdV-hACE2 system for nutrition studies with SARS-CoV-2. Future studies could also benefit from using 308 another mouse model, for example, hACE2 transgenic mice or a mouse-adapted strain of SARS-CoV-2. 309 controls. From study 1, two mice each from infected and uninfected groups were euthanized at 4-dpi and remaining at 10-dpi. In 320 study 2, four mice from infected and two from uninfected groups were euthanized at 4-dpi and remaining at 10-dpi. Results are 321 indicative of data combined from these duplicate independent studies. infected lean and obese mice was compared at 4-dpi (**p-value for IFN-β=0.0042; *p-value for 328 and 10-dpi (**p-value for IFN-β=0.2824, unpaired t-test; **p-value for IFN-γ=0.0022, Mann-Whitney test). The study was 329 conducted as two independent experiments with n=6 for SARS-CoV-2 infected groups and n=4 for only AdV-transduced 330 controls. From study 1, two mice each from infected and uninfected groups were euthanized at 4-dpi and remaining at 10-dpi. In 331 study 2, four mice from infected and two from uninfected groups were euthanized at 4-dpi and remaining at 10-dpi. Results are 332 indicative of data combined from these duplicate independent studies. infected groups (lean-AdV vs lean SARS-CoV-2; obese-AdV vs obese SARS-CoV-2; and between lean and obese SARS-CoV-2) 345 at 4-dpi between SARS-CoV-2 infected lean and obese mice (*p-value=0.0308, 2way ANOVA) and 10-dpi between obese-AdV 346 only and obese-SARS-CoV-2 groups (*p-value=0.0404, 2W-ANOVA). The study was conducted as two independent 347 experiments with n=6 for SARS-CoV-2 infected groups and n=4 for only AdV-transduced controls. From study 1, two mice each 348 from infected and uninfected groups were euthanized at 4-dpi and remaining at 10-dpi. In study 2, four mice from infected and 349 two from uninfected groups were euthanized at 4-dpi and remaining at 10-dpi. Results are indicative of data combined from these mice were compared (****p-value from 5-to 9-dpi<0.0001; **p-value from 9 to 15-dpi=0.002 to 0.007, 2way-ANOVA). Ethics approval and consent to participate 397 All animal handling protocols were approved by the Institutional Animal Care and Use Committee 398 (Protocol #20-060) at Virginia Tech. Consent to participate "not applicable" for this study. 399 

A major outbreak of severe acute respiratory syndrome in Hong Kong

Coronavirus as a possible cause of severe acute respiratory syndrome

ACE2 receptor expression and severe acute respiratory syndrome coronavirus 409 infection depend on differentiation of human airway epithelia

Middle East respiratory syndrome coronavirus infection in dromedary camels 413 in Saudi Arabia. mBio

Middle East respiratory syndrome coronavirus in bats, Saudi Arabia

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia

Clinical Characteristics of Coronavirus Disease 2019 in China

A pneumonia outbreak associated with a new coronavirus of probable bat origin. 422 Nature

Prognostic Tool for Early Clinical Decompensation

Risk factors for SARS-CoV-2 among patients in the Oxford Royal College of 427

General Practitioners Research and Surveillance Centre primary care network: a cross-sectional 428 study. The Lancet Infectious Diseases

Factors associated with hospital admission and critical illness among 5279 430 people with coronavirus disease

Middle East respiratory syndrome coronavirus: risk factors and determinants of 433 primary, household, and nosocomial transmission

Risk factors for severe outcomes following 2009 influenza A (H1N1) 435 infection: a global pooled analysis

Impaired wound healing predisposes obese mice to severe influenza virus 437 infection. The Journal of infectious diseases

National diabetes statistics report, 2020 : estimates of diabetes and its burden in the United 441 States

The Definition and Prevalence of Obesity and Metabolic Syndrome

Diet-induced obese mice have increased mortality and altered immune 445 responses when infected with influenza virus

Obesity-Related Microenvironment Promotes Emergence of Virulent Influenza 447 Virus Strains. mBio

Functional assessment of cell entry and receptor usage for 449 SARS-CoV-2 and other lineage B betacoronaviruses

Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based 451 on Decade-Long Structural Studies of SARS Coronavirus

Lethal infection of K18-hACE2 mice infected with severe acute respiratory 453 syndrome coronavirus

Mice transgenic for human angiotensin-converting enzyme 2 provide a model 455 for SARS coronavirus infection

Differential Virological and Immunological Outcome of Severe Acute 457 Respiratory Syndrome Coronavirus Infection in Susceptible and Resistant Transgenic Mice 458

Expressing Human Angiotensin-Converting Enzyme 2

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures. 463 Nature

A Mouse-Adapted SARS-CoV-2 Induces Acute Lung Injury and Mortality in 465 Standard Laboratory Mice

A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by 467 Neutralizing Antibodies

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and 469 Treatment

The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and 471 aged mice and is neutralized by convalescent and post-vaccination human sera. medRxiv

Pathogenesis and transmission of SARS-CoV-2 in golden hamsters

Oral SARS-CoV-2 Inoculation Establishes Subclinical Respiratory Infection with 475

Infection and Rapid Transmission of SARS-CoV-2 in Ferrets

SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over 479 more than one meter distance

Adenovirus targets transcriptional and posttranslational mechanisms to limit 482 gap junction function

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from 484 disease in a small animal model

Analysis of relative gene expression data using real-time 486 quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods

High Prevalence of Obesity in Severe Acute Respiratory Syndrome 488

Coronavirus-2 (SARS-CoV-2) Requiring Invasive Mechanical Ventilation

Risk factors for severe complications of the novel influenza A (H1N1): analysis 491 of patients hospitalized in Italy

An Analysis of 332 Fatalities Infected with Pandemic 2009 Influenza A 493 (H1N1) in Argentina

Obesity and the risk and outcome of infection

Host nutritional status affects alphavirus virulence, transmission, and 497 evolution

Nutritional status impacts dengue virus infection in mice

Impaired type I interferon activity and inflammatory responses in severe 501 COVID-19 patients

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

COVID-19: consider cytokine storm syndromes and immunosuppression

Immune cell profiling of COVID-19 patients in the recovery stage by single-cell 507 sequencing

COVID-19 severity correlates with airway epithelium-immune cell interactions 509 identified by single-cell analysis

Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease 511 severity predictors. Emerging Microbes & Infections

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

IFN-γ is an independent risk factor associated with mortality in patients with 515 moderate and severe COVID-19 infection

Antiviral Activity of Type I, II, and III Interferons Counterbalances ACE2 517 Inducibility and Restricts SARS-CoV-2. mBio

Transfer of Genes to Humans: Early Lessons and Obstacles to Success

A genetically engineered adenovirus vector targeted to CD40 mediates 521 transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo. 522 Vaccine

Long-term humoral and cellular immunity induced by a single immunization 524 with replication-defective adenovirus recombinant vector

Cellular and humoral immune responses to viral antigens create barriers to lung-527 directed gene therapy with recombinant adenoviruses

Immediate inflammatory responses to adenovirus-mediated gene transfer 530 in rat salivary glands

Innate immune mechanisms dominate elimination of adenoviral vectors 532 following in vivo administration

Acute hepatotoxicity of oncolytic adenoviruses in mouse models is associated 534 with expression of wild-type E1a and induction of TNF-α. Virology

Evaluation of toxicity from high-dose systemic administration of 536 recombinant adenovirus vector in vector-naive and pre-immunized mice

Activation of innate immunity in nonhuman primates following intraportal 539 administration of adenoviral vectors

Developing Covid-19 Vaccines at Pandemic Speed

Dietary obesity in nine inbred mouse strains