Mrs. DISHANTI MRISTA.
(Masters in pharmaceutics, Patuakhali Science and Technology University of Bangladesh)
VOL .03 ISSUE 11
There’s an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent a perfect strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods like formalin, heat, ultraviolet illumination, and gamma rays cause structural alterations in vaccines that cause reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2- mediated immune pathology. We’ve got evaluated the potential of a visual ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvant. Specifically, we demonstrate that vaccination of mice with the laser-inactivated H1N1 influenza virus at a couple of 10-fold lower doses than that required using conventional formalin-inactivated influenza vaccines ends up in protection against lethal H1N1 challenge in mice.
The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay Introduction Emerging viral pathogens are a continuing and prominent threat to human health worldwide, as evidenced by the recent outbreak of severe acute respiratory syndrome (SARS), geographic region respiratory syndrome.
Additionally, there’s a persistent risk of engineered viruses derived from bioterrorism. The foremost logical and cost-effective strategy to shield the human population from these emerging viral diseases is thru immunization. Thus, there’s a dire need for rapid methods to develop vaccines in response to new viral pathogens. Whole inactivated virus (WIV) vaccines represent a perfect strategy for this purpose. In contrast to subunit or recombinant vector vaccines, WIV vaccines circumvent the requirement to spot relevant antigens and may be quickly produced from purified viruses using chemical or physical inactivation methods. WIV vaccines contain many of the immunogenic epitopes and immune-stimulatory components (such as toll-like receptor ligands) that are needed for an efficient virus-specific immunologic response.
Emerging viral pathogens are a continuing and prominent threat to human health worldwide, as evidenced by the recent outbreak of severe acute respiratory syndrome (SARS), Near East respiratory syndrome (MERS) coronaviruses, and novel influenza strains with pandemic potential. additionally, there’s a persistent risk of engineered viruses derived from bioterrorism. the foremost logical and cost-effective strategy to shield the human popula- tion from these emerging viral diseases is thru immunization. Thus, there’s a dire need for rapid methods to develop vaccines in response to new viral pathogens. Whole inactivated virus (WIV) vaccines represent a perfect strategy for this purpose. In contrast to subunit or recombinant vector vaccines, WIV vaccines circumvent the requirement to spot relevant antigens and might be quickly produced from purified virus using chemical or physical inactivation methods. WIV vaccines contain many of the immunogenic epitopes and immunostimulatory components (such as toll-like receptor ligands) that are needed for an efficient virus-specific immune reaction. WIV vaccine production. actinic radiation (in the wavelength range of 400 to 700 nm) lacks the photon energy to disrupt covalent bonds in biological macromolecules like viruses; therefore, USP laser irradiation mustn’t generate a Th2 response inducing carbonyl groups in proteins. In the absence of chromospheres, visible radiation shows negligible. intrinsic absorption by proteins and nucleic acids; thus, USP laser irradiation mustn’t denature structural B-cell epitopes through heating. . Furthermore, the USP laser method doesn’t involve introducing any potentially toxic or carcinogenic chemicals during treatment and would alleviate public concerns in that regard. These rationales make the USP laser treatment a lovely potential method to come up with safe and effective WIV vaccines. In this paper, we demonstrate that immunization of mice with the USP laser-inactivated whole inactivated H1N1 influenza virus at a couple of 10-fold lower doses than that required by conventionally formalin-inactivated H1N1 vaccine leads to protection against lethal-dose H1N1 influenza challenge. Additionally, we employed a neutralization assay to verify the presence of neutralizing antibodies.
Furthermore, we’ve evaluated the consequences of USP laser–treatment on a model protein—bovine albumin (BSA). we’ve got found that, in contrast to traditional pathogen inactivation methods, the USP laser treatment failed to generate Th2 response-inducing carbonyl groups in BSA protein. Therefore, USP laser treatment may be a novel and a gorgeous potential strategy to come up with WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques.
MATERIALS AND METHOD
The mice were kept in an exceedingly pathogen-free environment at Johns Hopkins University (Baltimore, Maryland). Cells Madin Darby canine kidney (MDCK) cells, obtained from ATCC (Manassas, Virginia), were used for dead vitro assays. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with L-glutamine and 10% FBS at 37°C with 5% CO2.
Virus The strain of virus utilized in this study was A/PR/8/34 (H1N1) from ATCC (Manassas, Virginia).
The remaining virus particles were then concentrated by centrifugation at 118;000 × g for 1 h at 4°C through a 20% sucrose cushion in phosphate-buffered saline (PBS). The virus was stored in aliquots at ?80°C. The titer of the virus was measured by the tissue culture infectious dose-50 (TCID50) assay.17 For the TCID50 assay, MDCK cells were plated on a 96-well plate. The virus was added in 10-fold dilutions using infection media (DMEM with 4?g?mL N-acetylated trypsin and 0.03% BSA) for every successive row of wells. The plates were stored in an incubator at 37°C and 5% CO2. After 4 days, formaldehyde was added to repair the cells and naphthol blue-black was added to stain the fixed cells. The plates were washed then the five hundred infectious dose was calculated using the Reed–Muench method.18 Laser Irradiation The excitation laser source employed during this work was a diode-pumped mode-locked Ti-sapphire laser. The laser produced a continual train of 65 fs pulses at a repetition rate of 80 MHz.14,15,19 The output of the second-harmonic generation of the Ti-sapphire laser was accustomed to irradiate the sample. The excitation laser was chosen to work at a wavelength of 415 nm and with a median power of 140 MW.
A compound lens of focal length 7.5 cm was wont to focus the light beam into the sample area. so as to facilitate the interaction of the laser with the influenza virus particles, which were placed inside a Pyrex cuvette with solution, a magnetic stirrer was founded so virions would enter the laser-focused volume. All the laser-irradiated influenza samples contained approximately 2 × 108 TCID50?ml virus and had a volume of about 200 ?l. the standard exposure time of the sample to laser irradiation was about 8 h.
The inactivated virus was stored in aliquots at?80°C to be used in subsequent vaccination experiments. Hemagglutination Assay Live and therefore the USP laser-inactivated virus preparations were serially twofold-diluted during a 100 ml volume on a 96-well microtitre plate. A 0.5% chicken erythrocyte suspension was added to all or any wells and plates were incubated for 30 min on ice. Control mice didn’t receive vaccines. The mice in both groups maintained a uniform weight before administration of the challenge dose. period of time after the last vaccination, the mice were challenged with a dose (6 × 102 TCID50) of H1N1, and therefore the weight of the mice was daily monitored. Mice losing over 25% of their weight were considered to own reached the experimental endpoint and were euthanized. Detection of cellular surface CD8a and intracellular IFN-? was performed using flow cytometry as previously described.23 Briefly, the cells were incubated overnight with 1 ?g?ml of GolgiPlug (BD Pharmingen) in the presence of two ?g?ml of NP peptide. After washing withFACScan buffer, the cells were stained with -conjugated anti-mouse CD8a antibody. Gating was performed on the lymphocyte area. Micro-neutralization Assay Blood was collected from the tail vein of vaccinated (n 5) or unvaccinated (n 4) mice 2 weeks after the last vaccination. The serum was stored in aliquots at 4°C.
After the serum was collected, the mice were tested for H1N1-specific neutralizing antibodies as follows. 2 × 104 MDCK cells were plated in each well of a 96-well plate. Serum was diluted with infection media (DMEM with 4 ?g?mL N-acetylated trypsin and 0.03% BSA) to 1:100 and added to the primary row of wells containing the MDCK cells. After thorough mixing of the good contents, 25 ?l of the primary row’s wells were added to 25 ?l of infection media within the next row a relentless H1N1 concentration of 1.75 × 105 TCID50?well was used for every plate.
The virus and serum were incubated at 25°C for two hours so added to the 96-well plate with MDCK cells. The plates were stored for 3 nights in an incubator at 37°C and 5% CO2. Formaldehyde and naphthol blue-black was added to visualize the results of the reaction as within the TCID50 assay.
This assay was repeated 3 times. Neutralization titers were calculated using the Reed Muench method. The inverse of the very best dilution at which 50% protection was achieved make up my mind to be the neutralization titer of the serum. groups in protein;4,10,11,27 these carbonyl groups are successively inducers of harmful and undesirable T helper type 2 responses.4,5 to see whether laser treatment generates protein carbonylation, we used a colorimetric 2,4-dinitrophenyl- hydrazine (DNPH)-based assay to quantitate carbonyl content in laser-treated BSA.
Figure 5 shows that there’s no significant increase in carbonyl content in laser-treated BSA samples relative to untreated BSA. These results demonstrate that USP laser treatment generates fewer carbonyl groups in protein antigens compared with conventional pathogen inactivation methods, reducing the danger of detrimental vaccine-elicited Th2 responses. First, we’ve estimated the amount of UVC photons that may be produced at the laser power densities utilized in our experiments. The two-photon absorptance for the H1N1 flu virus isn’t available within the literature; however, if we assume that the viruses are giant molecules whose two-photon absorptance at 415 nm is like that of a typical molecule, 28 then the amount of UVC photons at 208 nm generated under our experimental conditions was estimated to be of the order of 0.1/ second. Because the laser exposure time is 8 h, the quantity of generated UVC photons is about 3000 for the overall laser exposure time. On the opposite hand, the whole number of H1N1 viral particles within the laser-irradiated sample is about 200 million; since one UVC photon at 208 nm can inactivate just one viral particle, the worth of generated UVC photons of about 3000 during the full laser exposure time is simply too small to account for the whole inactivation of 200 million viral particles observed in our experiments. Second, we’ve tried to detect the UVC photons which could be generated in our laser experiments (here, the glass vial is replaced by an artificially fused silica vial) by employing a UV spectrometer with a photon-counting system. Within our experimental uncertainty of 0.5 photons? second, we didn’t detect any of the UVC photons at 208 nm. This finding is in keeping with our estimation stated above. additionally, we note that the coefficient of absorption of water at 208 nm is about 0.05 cm?1, which suggests that the effect of water absorption at 208 nm is negligible and can’t be the explanation for not detecting the UVC photon. Therefore, the observed complete inactivation for H1N1 flu virus observed in our experiments can’t be thanks to UVC photons generated by two-photon absorption. When the laser intensity is sufficiently large, the excited amplitude of vibration on the capsid can become extremely large, resulting in the breaking of weak links like hydrogen bonds and hydrophobic contacts within the capsid of the virus. This causes the capsid to disintegrate into subunits. The virus becomes inactivated due to the loss of integrity of its capsid. On the opposite hand, it’s been demonstrated that enveloped viruses like murine cytomegalovirus (MCMV) are inactivated by a visual USP laser via the ISRS process likewise but through a unique route/pathway.19 The USP laser pulses excite Raman-active, However, if the concentration of proteins is extremely high, like within the case of proteins confined within the capsid of any will form hydrogen bonds and hydrophobic contacts with other proteins nearby, resulting in the aggregation of proteins.
Aggregation between capsid proteins and tegument proteins has been found to be the reason behind the inactivation of an enveloped virus. All influenza viruses are enveloped viruses. Therefore, we believe that the foremost likely inactivation mechanism for the H1NI influenza virus inactivated by the visible USP laser studied here is that the ISRS process. the underlining inactivation mechanism) of the USP laser irradiation on these two enveloped viruses are identical. To give a perspective on the vaccine potency reported in our current study, it’s illustrative to check the vaccine dosages required for defense against lethal challenge in mice using conventional or laser-inactivated H1N1 influenza vaccines. Among conventional methods including heat, formalin, beta propiolactone, and detergents, formalin was found to own the best preservation of influenza vaccine antigens.
Figure 6(a) shows one control group vaccinated with two doses of conventional formalin- inactivated H1N1 vaccine at ?2.76 × 106 pfu?dose, which was the dose of the USP laser-inactivated vaccine that we used for defense against the challenge. Figure 6(b) shows the opposite control group vaccinated with two doses of conventional formalin-inactivated H1N1 vaccine at ?2.76 × 107 pfu?dose. The mice of the control group in Fig. 6(a) all died (the weights decreased quite 30% of their initial ones) 10 days after challenging; whereas the mice of the control group in Fig. 6(b) achieved 75% protection. On the opposite hand, with the visible USP laser irradiation method, we employed two doses of USP laser-inactivated vaccine at ?2.76 × 106 pfu?dose, which may be a 10-fold lower dose relative to the formalin-inactivated vaccine, to attain 87.5% protection. Therefore, these results indicate that the USP laser-inactivated vaccine is significantly more efficient than the vaccine prepared by the standard formalin-inactivated vaccine. The relatively high potency of the USP laser-inactivated influenza virus vaccine is partly attributed to the very fact that the visible USP laser irradiation has minimal effects on the structure of proteins. The circular dichroism (CD) spectrum of BSA protein measured before and after the USP laser irradiation could be an example. The CD spectrum is incredibly sensitive to the secondary structure of proteins. it’s been shown that, within experimental uncertainty, there’s no change within the CD spectrum of BSA protein before and after USP laser irradiation.15 These spectroscopic results are in line with our hemagglutination activity results We note that the USP laser-inactivated influenza vaccine may generate heterosubtypic immunity, which is that the aim of current efforts to style universal influenza vaccines. The CTL response could be a key mechanism for improved heterosubtypic protection against influenza because CTLs are shown to be specific for epitopes that are conserved among viral subtypes.29 These results suggest that the laser-inactivated influenza vaccine has the potential to get cross-protection against multiple strains and address the difficulty of viral mutation. The presence of carbonyl groups in vaccine antigens has been linked to the induction of undesirable and potentially deleterious Th2-mediated immunopathology.4,5 Many inactivation techniques including UV and nonparticulate radiation are potent inducers of protein carbonylation.4,10,11,27 We note that although formaldehyde isn’t an oxidizer and can’t produce oxidative damage in an exceedingly cell-free system, the carbonyl groups introduced into proteins by formaldehyde within the formalin inactivation technique have something in common with the carbonyl groups introduced by protein oxidation. In contrast to those techniques, visible USP lasers lack the energy to disrupt covalent structures in proteins. Therefore, we reasoned that USP laser treatment wouldn’t cause protein carbonylation. The experimental leads to Fig. 5 confirm this. These data demonstrate that USP laser treatment doesn’t generate significant levels of carbonyl groups in protein antigens compared with conventional pathogen inactivation methods, reducing the danger of detrimental vaccine-elicited Th2 responses. administered to patients, as was seen in penicillin allergies30 and certain chemically treated blood products.31 In contrast, the USP laser inactivates enveloped influenza virus through the disruption of weak, noncovalent hydrogen bonds and hydrophobic contacts within the virion, resulting in the aggregation of capsid and tegument proteins.
In summary, we’ve got demonstrated a unique USP laser irradiation method for the assembly of safe and potent WIV vaccines. We envision that the longer term of pathogen inactivation technologies will favor chemical-free methods that concentrate on properties specific to pathogens while preserving desirable components of the treated product, resulting in improved safety profiles. The USP laser irradiation method we’ve presented during this report is one such potential technology.
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Shaw-Wei David Tsen,a Nisha Donthi,b Victor La,b Wen-Han Hsieh,b Yen-Der Li,c Jayne Knoff,b Alexander Chen,b Tzyy-Choou Wu,b,d,e,f Chien-Fu Hung,b,f,* Samuel Achilefu,a,g,h and Kong-Thon Tseni,* aWashington University School of Medicine, Department of Radiology, St. Louis, Missouri 63110, United States
bJohns Hopkins Medical Institutions, Department of Pathology, Baltimore, Maryland 21231, United States
cNational Taiwan University, College of Medicine, Taipei 10617, Taiwan
dJohns Hopkins Medical Institutions, Department of Obstetrics and Gynecology, Baltimore, Maryland 21231, United States
eJohns Hopkins Medical Institutions, Department of Molecular Microbiology and Immunology, Baltimore, Maryland 21231, United States
fJohns Hopkins Medical Institutions, Department of Oncology, Baltimore, Maryland 21231, United States
gWashington University School of Medicine, Department of Biochemistry and Molecular Biophysics, St. Louis, Missouri 63110, United States
hWashington University School of Medicine, Department of Biomedical Engineering, St. Louis, Missouri 63110, United States
iArizona State University, Department of Physics and Center for Biophysics, Tempe, Arizona 85287, United States
Chemical-free inactivated whole influenza virus vaccine prepared by ultrashort pulsed laser treatment; published online Nov. 25, 2014.