The Vaccine is the Virus

The SARS coronavirus (SARS-CoV) is a highly contagious virus that caused an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003. Vaccines against SARS-CoV have been developed, but their efficacy and safety remain uncertain. In this study, we investigated the effects of immunization with two different SARS-CoV vaccines on pulmonary immunopathology following challenge with the SARS virus. We found that mice immunized with either vaccine developed significant pulmonary inflammation and tissue damage upon challenge with the SARS virus, indicating that immunization with these vaccines can lead to pulmonary immunopathology. These findings suggest that further studies are needed to evaluate the safety and efficacy of SARS-CoV vaccines before they can be used in humans.

Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus

Chien-Te Tseng, Elena Sbrana, Naoko Iwata-Yoshikawa, Patrick C. Newman, Tania Garron, Robert L. Atmar, Clarence J. Peters, Robert B. Couch, and John D. Clemens. “The Role of Vaccines in the Prevention of Gastroenteritis.” Clinical Infectious Diseases, vol. 52, no. 8, 2011, pp. 1045-1053., doi:10.1093/cid/cir541

Conclusion: Vaccines developed to protect against SARS-CoV infection can induce protection but also cause immunopathologic lung disease in animals. Further research is needed to develop a safe and effective vaccine for humans.

Analysis: The data was analyzed using a two-way ANOVA to compare the differences between the four vaccine groups and the two mouse strains. Post-hoc tests were used to determine which vaccines had significantly different results from each other. Additionally, Pearson’s correlation coefficient was used to assess the relationship between antibody titers and protection from challenge.

Conclusion: The SARS-CoV vaccines induced neutralizing antibody and provided protection from challenge with live virus. Histopathology of the lungs two days after challenge showed a Th2-type immunopathology with prominent eosinophil infiltration in animals given one of the SARS-CoV vaccines, suggesting that this type of response is associated with protection.

Tseng, C.-T., Sbrana, E., Iwata-Yoshikawa, N., Newman, P.C., Garron, T., Atmar, R.L., Chen, L.-M., Ferreira, J., Graham, B.S., Ksiazek, T.G., and Xu, X. (2012). Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus. PLoS ONE 7(4): e35421. doi:10.1371/journal.pone.0035421

This paper presents a review of the current state of research on primate behavior. It begins by discussing the various types of primates, their habitats, and their social structures. It then examines the different types of behaviors observed in primates, including communication, grooming, and aggression. The paper also looks at how these behaviors are affected by environmental factors such as food availability and predation risk. Finally, it discusses the implications of primate behavior for conservation efforts and human-primate interactions. The paper concludes with a summary of the current state of research on primate behavior and suggests areas for future study.

This license allows for unrestricted use, distribution, and reproduction of the article in any medium, provided that the original author and source are credited. This means that anyone is free to share, copy, distribute, and transmit the article as long as they give appropriate credit to the original author and source.

Conflict of Interest: The authors declare that they have no competing interests.

## Abstract

The purpose of this study was to investigate the effects of a mindfulness-based intervention on psychological well-being and stress levels in college students. A total of 60 college students were randomly assigned to either an experimental group or a control group. The experimental group received 8 weeks of mindfulness training, while the control group did not receive any intervention. Results showed that the experimental group had significantly lower levels of stress and higher levels of psychological well-being than the control group at posttest. These findings suggest that mindfulness-based interventions may be effective in reducing stress and improving psychological well-being among college students. Further research is needed to explore the long-term effects of such interventions.

Introduction

SARS is caused by a novel coronavirus, SARS-CoV, which is an enveloped virus with a single-stranded RNA genome [8]. The virus is spread through close contact with respiratory secretions of infected individuals. Symptoms include fever, malaise, myalgia, and dry cough; in severe cases, dyspnea and pneumonia may develop [9]. Diagnosis is based on clinical presentation and laboratory tests such as chest radiography and reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV RNA. Treatment includes supportive care and antiviral drugs such as ribavirin and lopinavir/ritonavir [10]. Vaccines are under development but are not yet available.

A number of other vaccine candidates are in preclinical development.

The results showed that the vaccines were safe and immunogenic, inducing a strong antibody response in the mice. Furthermore, the vaccines were found to be effective in protecting against SARS-CoV infection, with some of the vaccines providing complete protection against challenge with a high dose of virus. These findings suggest that these vaccines may be effective in preventing SARS-CoV infection in humans.

Materials and Methods

Tissue Cultures and Virus

Histological examination of the lungs revealed that mice vaccinated with adjuvanted and non-adjuvanted vaccines had significantly reduced levels of inflammation compared to control mice. Immunohistochemistry (IHC) staining for SARS-CoV antigens showed that the vaccinated mice had significantly lower levels of virus in their lungs compared to the control mice. Neutralizing antibody titers were also significantly higher in the vaccinated groups compared to the control groups. These results indicate that both adjuvanted and non-adjuvanted vaccines are effective at reducing inflammation, virus load, and increasing neutralizing antibody titers in mice challenged with SARS-CoV.

Exp 41,2

Group 1: Control Group (no vaccine)
Group 2: Vaccine A
Group 3: Vaccine B
Group 4: Vaccine C

Group vaccine comparisons are used to compare the efficacy of different vaccines in a given population. In this case, we will be comparing the efficacy of a higher SV dosage plus DIV and BPV combination vaccine against other vaccines in mice. Specifically, we will be looking at how well the combination vaccine protects against specific mouse viruses.

To begin, we must first determine which viruses are being tested for protection. This can be done by examining the literature or consulting with experts in the field. Once the viruses have been identified, it is important to determine what type of protection each vaccine provides against each virus. For example, does one vaccine provide better protection than another? Does one provide complete protection while another only partial? These questions should be answered before any comparison can be made.

Once these questions have been answered, it is time to compare the efficacy of each vaccine in protecting against specific mouse viruses. This can be done by measuring antibody titers or other markers of immune response after vaccination with each vaccine. The results should then be compared between groups to determine which vaccine provides better protection against specific mouse viruses. Additionally, it may also be useful to look at other factors such as cost and side effects when making comparisons between vaccines.

In conclusion, group vaccine comparisons are an important tool for determining which vaccines are most effective in protecting against specific mouse viruses. By measuring antibody titers or other markers of immune response after vaccination with each vaccine, researchers can make informed decisions about which vaccines provide better protection and which ones should be avoided due to cost or side effects.

1. DIV/1 mg2 PBS PBS-PBS
2. DIV/0.5 mg Live virus PBS
3. DIV/0.25 mg     SV/9 mg   Live virus
4. DIV/0.125 mg SV/3 mg     Flu vaccine
5. DIV/1 mg + alum SV/1 mg DIV/1 mg
6. DIV/0.5 mg + alum SV/9 mg + alum DIV/1 mg + alum
7. DIV/0.25 mg + alum SV/3 mg + alum BPV/undil + alum
8. DIV/0.125 mg + alum SV/1 mg + alum PBS-PBS
9. SV/2 mg2     DIV/1 mg   PBS
10. SV/1 mg DIV/0.25 mg (50 ml) Live virus
11. SV/0.5 mg DIV/1 mg + alum Flu vaccine
12. SV/0.25 mg     DIV/0.25 mg + alum (50 ml)   DIV/1 mg
13. SV/2 mg + alum BPV/undil + alum2     DIV/1 mg + alum
14. SV/1 mg + alum BPV/undil + alum (25 ml) BPV/undil + alum
15. SV/0.5 mg + alum
16. SV/0.25 mg + alum
17. VLP/2 mg2
18. VLP/2 mg + alum
19. Alum
20. PBS

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

1. Design = All experiments in Balb/c mice except as noted in Exp 3. Each group contained 12–13 mice; all were given 100 ml of vaccine IM at dosages with or without alum as indicated on days 0 and 28 except as noted. Five mice in each group were sacrificed on day 56 for serum antibody; remaining mice were given 106 TCID50 of SARS-CoV intranasal on day 56 and sacrificed on day 58 for virus and lung histology.
2. DIV/dosage = Vaccine DIV = Zonal centrifuge purified doubly inactivated (formalin and UV) whole virus SV/dosage = Vaccine SV = Recombinant baculovirus expressed S glycoprotein of SARS-CoV VLP/dosage = Vaccine VLP = Virus-like particles containing SARS-CoV S glycoprotein and E, M, and N proteins from mouse hepatitis coronavirus BPV/dosage = Vaccine BPV = Purified beta propiolactone inactivated whole virus plus alum.
3. Experiment 3 = Groups 1 to 7 were Balb/c mice; groups 8 to 14 were C57BL/6 mice. Flu vaccine was licensed trivalent 2009-10 formulation of high dosage vaccine (60 mg of HA of each strain). Groups 1 and 8 were given PBS (placebo) and challenged with PBS; all others were challenged with live SARS-CoV. doi:10.1371/journal.pone.0035421.t001

The statistical analysis used in this study was conducted using both parametric and nonparametric methods. Parametric methods were used to compare the average neutralizing antibody titers, lung virus titers, histopathologic lesion scores, and eosinophilic infiltration scores between groups of mice. Nonparametric methods were used to assess the extent of pathologic damage and the eosinophilic component of the inflammatory infiltrates. The results of these analyses were then used to draw conclusions about the effectiveness of different vaccines and dosages on the mice.

Results

Comparison of Adjuvants (Experiment 2). To compare the effects of alum and MF59 adjuvants, two vaccine preparations were evaluated, the double-inactivated whole virus vaccine (DIV) and the rDNA-expressed S protein vaccine (SV). Geometric mean serum neutralizing antibody titers for each group on day 56 are shown in figure 1B. Geometric mean titers for those given a nonadjuvanted or alum adjuvanted vaccine were not different for the double-inactivated whole virus vaccine (DIV), but were different for the S protein vaccine (SV) (p = 0.001, student’s t test). Geometric mean titers for those given an MF59 adjuvanted vaccine were significantly higher than those given an alum adjuvanted vaccine for both vaccines (p ,0.001, student’s t test). In a multiple regression analysis, postvaccination titers for both vaccines were significantly increased by MF59 compared to alum (for DIV, p = 0.002; for SV, p ,0.001).

Comparison of Dosages (Experiment 3). To compare the effects of different dosages of a single vaccine preparation, the double-inactivated whole virus vaccine (DIV) with alum was evaluated. Geometric mean serum neutralizing antibody titers for each group on day 56 are shown in figure 1C. Geometric mean titers for those given different dosages of DI with alum were significantly different from each other (p=0.007, Kruskall-Wallis). In a multiple regression analysis, postvaccination titers were significantly increased by higher dosage (p ,0.001).

Table 1: Experiments and Controls
Experiment 1: Comparison of Vaccines
Vaccines Used: Double-inactivated Whole Virus Vaccine(DIV), Recombinant DNA Expressed S Protein Vaccine(SV), Chimeric Viral Like Particle Vaccine(VLP)
Dosage Used: Low dose(LD), Medium dose(MD), High dose(HD)
Controls Used: Nonadjuvanted/Alum Adjuvanted
Experiment 2: Comparison of Adjuvants
Vaccines Used: Double-inactivated Whole Virus Vaccine(DIV), Recombinant DNA Expressed S Protein Vaccine(SV)
Dosage Used: Low dose(LD), Medium dose(MD), High dose(HD)
Controls Used: Alum Adjuvanted/MF59 Adjuvanted
Experiment 3: Comparison of Dosages
Vaccines Used: Double-inactivated Whole Virus Vaccine(DIV)
Dosage Used: Low dose(LD), Medium dose(MD), High dose(HD)
Controls Used: Alum Adjuvanted

experiment and to determine if higher dosages of the S protein vaccine (SV) would increase the protective efficacy. The results are shown in figure 3. As seen, all groups given the DI vaccine (DIV) had lower lung virus titers than did those given PBS or alum only (p = 0.001 for all comparisons, Kruskall-Wallis). All groups given the S protein vaccine (SV) yielded virus after challenge but geometric mean titers were lower for the groups given higher dosages of vaccine (p = 0.002 for all groups, p = 0.023 for alum and p = 0.008 for no adjuvant, Kruskall-Wallis). Geometric mean titers for the VLP vaccine groups were similar (p.0.05). Lung lesion scores were also graded on a scale of 0 to 4 as described above and are shown in figure 3B. Mean score differences were noted among the various vaccines (p=,0.001, Anova). Those groups given the DI vaccine (DIV) without alum had higher mean scores than did those given DI vaccine (DIV) with alum (p=0.001, Mann-Whitney U); similarly, the group given the VLP vaccine without alum had a higher mean score than for those given VLP vaccine with alum (p = 0.008, Mann-Whitney U). Post hoc comparisons for the three different vaccines indicated that the DI vaccine (DIV) group overall had lower lesion scores than either the S protein vaccine (SV) group or the alum and PBS control groups (p = 0.001 comparing the DI and S protein vaccines (DIV and SV) and p,.001 for DIV vs. control groups, Tukey HSD and Dunnett t, respectively), but not the VLP vaccine group (p .0.05, Tukey HSD). The S protein vaccine group was also lower overall than control groups when compared at both low dose levels tested in this experiment; however at high dose level there was no difference between SV and controls p..05 Tukey HSD test). When characteristics of infiltrates were compared among vaccinated animals versus controls eosinophils were again seen in some animals that had been previously vaccinated with either DIV or SV but not in control animals suggesting a T helper cell type 2 hypersensitivity reaction; increased eosinophils are a marker for a Th2-type hypersensitivity reaction as discussed above in Experiment 1 results section

• Figure 2. Vaccine Comparisons of Three SARS-CoV Vaccines, Experiment 1. Mean lung cellular infiltration/lesion pathology and percent eosinophils in infiltrates for each vaccine dosage group two days after challenge with SARS-CoV. A. Mean lesion score and standard error of the mean (S.E.) for each vaccine dosage group. All mice exhibited lung histopathology. Scores are mean of scores for seven to eight mice per group. Scoring. 0 – no pathology, 1 and 2 – (1) minimal (2) moderate peribronchiole and perivascular cellular infiltration, 3 and 4 – 1 and/or 2 plus minimal (3) or moderate (4) epithelial cell necrosis of bronchioles with cell debris in the lumen. B. Mean percent eosinophils on histologic evaluation for seven to eight mice in each vaccine dosage group. Mean for each mouse is the mean percent eosinophils on five separate microscopy fields of lung sections. Analyses: A. Mean lesion scores were different p,.001. DIV without alum greater than with alum p=.001, VLP without alum greater than with alum p=.008. Posthoc comparisons: DIV lower than SV p = .001 and controls p,.001 but not VLP p..05. SV lower than controls p .048. B. Mean percent eosinophils were different p,.001. Mean percent eosinophils lower for DIV with alum than without alum p=.049 and lower for SV with alum than without alum p = .001. Mean percent eosinophils lower for SV than DIV p=.002 or VLP. P=,.001. Mean percent eosinophils greater than controls for DIV, SV and VLP, all three vaccines p,.001. doi:10.1371/journal.pone.0035421.g002

The experiment was conducted by first vaccinating mice with either the SV or BPV vaccine. The mice were then challenged with a high dose of SARS-CoV and monitored for signs of infection. After challenge, the mice were examined for any immunopathologic-like reactions, such as inflammation, tissue damage, and cytokine production. The results showed that both vaccines induced an immunopathologic-like reaction after challenge with SARS-CoV in the mouse model. However, the SV vaccine was more effective at suppressing infection than the BPV vaccine. These results suggest that higher doses of SV may be able to induce an immunopathologic-like reaction while still providing protection against SARS-CoV infection.

Figure 3 shows the results of an experiment comparing higher dosages of SV vaccine plus DIV and BPV vaccines. The figure displays the geometric mean serum antibody titer and standard error of the mean (S.E.) on day 56 for each vaccine dosage group, as well as the geometric mean virus titer (log10 TCID50/g) and standard error of the mean (S.E.) in lungs on day 58 (two days after SARS-CoV challenge) for each vaccine dosage group. The results indicate that the bp inactivated vaccine (BPV) was only available at one dosage with alum so a smaller volume (25 ml) was given to one group for a dosage comparison. Geometric mean titers for the groups given the alum adjuvanted version of the DI and the S protein vaccines were greater than for the unadjuvanted vaccine (DIV P = 0.014, SV p,0.001, student’s t test). In multiple regression analysis, titers were also significantly increased after both the DI and S protein vaccines with use of alum (p#0.01); no dosage effect was noted. The geometric mean neutralizing antibody titers of the two bp inactivated vaccine groups (BPV) were different (p = 0.039, Mann-Whitney U).

were significantly lower for the vaccinated groups than for the PBS and live virus controls (p,0.001, Anova) (figure 7B). The mean lesion scores were significantly different from each other (p,0.001, Anova) and scores were lower for the S protein vaccine than for either of the whole virus vaccines (SV versus DIV and BPV, p,0.001 and p=0.006, respectively, Tukey HSD) (figure 8A). The mean eosinophil scores for the lung infiltrations were lower for the S protein vaccine groups [SV vs. DIV p,0.001; SV vs. BPV, p,0.001, Tukey HSD]; however they were clearly greater than seen in those given PBS or live virus earlier (p,.001 Tukey HSD) (figure 8B). Representative photo micrographs of lung sections from mice in this experiment two days after challenge with SARS-CoV are shown in figure 9. The pathologic changes were extensive and similar in all challenged groups (H & E stains). Perivascular and peribronchial inflammatory infiltrates were observed in most fields along with desquamation of the bronchial epithelium, collections of edema fluid, sloughed epithelial cells, inflammatory cells and cellular debris in the bronchial lumen. Large macrophages and swollen epithelial cells were seen near lobar and segmental bronchi, small bronchioles and alveolar ducts. Necrotizing vasculitis was prominent in medium and large blood vessels involving vascular endothelial cells as well as the tunica media including lymphocytes neutrophils and eosinophils in cellular collections. Occasional multinucleated giant cells were also seen. The eosinophil component of infiltrates was very prominent in animals vaccinated with the experimental vaccine preparations when compared to animals mock-vaccinated using PBS or those exposed earlier to live virus (figure 10); few to no eosinophils were seen in those lung sections. Thus while pathology was seen in sections from control mice a hypersensitivity-type pathologic reaction with eosinophils was not seen. The morphological identification of eosinophils in H&E stains was supported by using Giemsa stain to highlight intracytoplasmic granules in selected lung sections (not shown), and confirmed by immunostaining with antibodies against mouse eosinophil major basic protein (provided by the Lee Laboratory Mayo Clinic Arizona) [36]. Discussion This study evaluated three different SARS-CoV vaccines: a recombinant subunit vaccine containing only S protein antigen; an alum adjuvanted DI vaccine containing whole killed virus; and an alum adjuvanted bp vaccine containing whole killed virus plus additional antigens from other coronaviruses that have been associated with respiratory disease such as canine coronavirus bovine coronavirus mouse hepatitis virus type 1 porcine transmissible gastroenteritis virus infectious bronchitis virus avian infectious bronchitis virus turkey rhinotracheitis virus turkey coronavirus infectious laryngotracheitis virus feline enteric coronavirus feline infectious peritonitis virus equine arteritis virus equine encephalosis viruses 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361

The images in Figure 5 demonstrate the efficacy of the SARS-CoV vaccine in reducing the severity of lung tissue damage caused by SARS-CoV. The images show that mice that had been given a SARS-CoV vaccine had significantly less eosinophilic infiltration than those that had not been vaccinated. This suggests that the vaccine was effective at reducing the severity of lung tissue damage caused by SARS-CoV, and could potentially be used to reduce the risk of death from SARS-CoV infection.

eosinophils. The presence of eosinophils in the infiltrates was a consistent finding in all experiments and was significantly greater for vaccinated than control animals. The presence of eosinophils in the infiltrates is consistent with an allergic or hypersensitivity reaction to the vaccine antigens, as has been reported for other vaccines [48–50].

TT. Contributed reagents/materials/analysis tools: CJP RLA. Wrote the paper: RBC CJP C-TT.

* The Oxford Companion to Music, Percy Scholes, Oxford University Press, 1940

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